Pseudomonas

J Bacteriol, 1996 Sep, 178(18), 5508 - 12
Physiological changes and alk gene instability in Pseudomonas oleovorans during induction and expression of alk genes; Chen Q et al.; The alk genes of Pseudomonas oleovorans, which is able to metabolize alkanes and alkenes, are organized in alkST and alkBFGHJKL clusters, in which the expression of alkBFGHJKL is positively regulated by AlkS . Growth of the wild-type strain GPo1 and P . oleovorans GPo12 alk recombinants on octane resulted in changes of cellular physiology and morphology . These changes, which included lower growth rates and a reduction of the number of CFU due to filamentation, were also seen when the cells were grown on aqueous medium, and the alk genes were induced with dicyclopropylketone, a gratuitous inducer of the alk genes . These effects were seen only for recombinants carrying both alkST and alkBFGHJKL operons . Deletion of parts of either alkB or alkJ, which encode two major Alk proteins located in the cytoplasmic membrane, modified but did not eliminate the effects described above, suggesting that they were due to induction and expression of several alk genes . Continuous growth of the cells in the presence of dicyclopropylketone for about 10 generations led to inactivation, but not elimination, of the alk genes . This resulted in a return of the recombinants to normal physiology and growth.

Appl Environ Microbiol, 1996 Sep, 62(9), 3101 - 6
Toluene and ethylbenzene oxidation by purified naphthalene dioxygenase from Pseudomonas sp . strain NCIB 9816-4; Lee K et al.; Purified naphthalene dioxygenase (NDO) from Pseudomonas sp . strain NCIB 9816-4 oxidized toluene to benzyl alcohol and benzaldehyde by reactions involving benzylic monooxygenation and dioxygen-dependent alcohol oxidation, respectively . Xylene and nitrotoluene isomers were also oxidized to substituted benzyl alcohol and benzaldehyde derivatives . NDO oxidized ethylbenzene sequentially through (S)-1-phenethyl alcohol (77% enantiomeric excess) and acetophenone to 2-hydroxyacetophenone . In addition, NDO also oxidized ethylbenzene through styrene to (R)-1-phenyl-1,2-ethanediol (74% enantiomeric excess) by reactions involving desaturation and dihydroxylation, respectively . Isotope experiments with 18O2, H2 18O, and D2O suggest that 1-phenethyl alcohol is oxidized to acetophenone by a minor reaction involving desaturation followed by tautomerization . The major reaction in the conversion of 1-phenethyl alcohol and benzyl alcohol to acetophenone and benzaldehyde, respectively, probably involves monohydroxylation to form a gem-diol intermediate which stereospecifically loses the incoming hydroxyl group to leave the carbonyl product . These results are compared with similar reactions catalyzed by cytochrome P-450.

J Biol Chem, 1996 Aug 23, 271(34), 20322 - 30
Crystal structure of L-2-haloacid dehalogenase from Pseudomonas sp . YL . An alpha/beta hydrolase structure that is different from the alpha/beta hydrolase fold; Hisano T et al.; L-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of L-2-haloalkanoic acids to yield the corresponding D-2-hydroxyalkanoic acids . The crystal structure of the homodimeric enzyme from Pseudomonas sp . YL has been determined by a multiple isomorphous replacement method and refined at 2.5 A resolution to a crystallographic R-factor of 19.5% . The subunit consists of two structurally distinct domains: the core domain and the subdomain . The core domain has an alpha/beta structure formed by a six-stranded parallel beta-sheet flanked by five alpha-helices . The subdomain inserted into the core domain has a four helix bundle structure providing the greater part of the interface for dimer formation . There is an active site cavity between the domains . An experimentally identified nucleophilic residue, Asp-10, is located on a loop following the amino-terminal beta-strand in the core domain, and other functional residues, Thr-14, Arg-41, Ser-118, Lys-151, Tyr-157, Ser-175, Asn-177, and Asp-180, detected by a site-directed mutagenesis experiment, are arranged around the nucleophile in the active site . Although the enzyme is an alpha/beta-type hydrolase, it does not belong to the alpha/beta hydrolase fold family, from the viewpoint of the topological feature and the position of the nucleophile.

Biochemistry, 1996 Aug 20, 35(33), 10904 - 12
Metal-substrate interactions facilitate the catalytic activity of the bacterial phosphotriesterase; Hong SB et al.; The bacterial phosphotriesterase from Pseudomonas diminuta is a zinc metalloenzyme which catalyzes the hydrolysis of a variety of organophosphorus nerve agents with high efficiency . The active site of the enzyme consists of a coupled binuclear metal center embedded within a cluster of histidine residues . Potential protein-substrate interactions at the active site were probed by a systematic variation of metal identity, leaving group potential, phosphate host, and amino acid replacement . In order to determine the roles of these metal ions in binding and catalysis, the microscopic rate constants and kinetic parameters were obtained with various divalent cations . The divalent cations that were utilized in this investigation consisted of Co2+, Ni2+, Cd2+, Zn2+, Mn2+, and the mixed-metal Zn2+/Cd2+ hybrid . The leaving group potential and phosphate host were varied by altering the pKa of the departing substituted phenol or thiophenol in either a diethyl phosphate or a diethyl thiophosphate substrate . The Bronsted plots for the nonenzymatic hydroxide catalyzed hydrolysis of these substrates showed a linear dependence between the pseudo-first-order rate constant and the pKa of the leaving group . Enzymatic activities of the wild-type enzyme with these same substrates varied by over 7 orders of magnitude over the entire experimental pKa range (4.1-10.3), and the corresponding Bronsted plots were nonlinear . Those substrates with leaving groups with high pKa values were limited by the rate of bond cleavage while those substrates having leaving groups with low pKa values were limited by a conformational change or binding event . Thiophosphate substrates having leaving groups with high pKa values were better substrates than the corresponding phosphate analogues . These results are consistent with the direct coordination of one or both metal ions with the phosphoryl sulfur or oxygen atom of the substrate . A large dependence of the rate on the leaving group rules out the possibility of protonation of the leaving group or electrostatic interaction of the leaving group oxygen (or sulfur) with a metal ion or cationic group at the active site . The large differences in the size of the beta lg over the range of metal ions utilized by the enzyme indicate that the metal ions polarize the phosphoryl group and alter the structure of the transition state . The values of V/K(m) for the enzyme-catalyzed hydrolysis for a series of substituted thiophenol analogues were 10(2)-10(3)-fold smaller than those obtained for the hydrolysis of the corresponding phenolic substrates, suggesting that the bulkier sulfur substituent in the leaving group may induce conformational restrictions at the active site . With the zinc-substituted H201N mutant enzyme, there was a large decrease in the rate of phosphotriester hydrolysis but essentially no change in the rate of thiophosphotriester hydrolysis relative to the values observed for the zinc-substituted wild-type enzyme . These results suggest that a direct perturbation in the ligand structure of the binuclear metal center induces alterations in the mechanism of substrate hydrolysis.

Biosci Biotechnol Biochem, 1996 Aug, 60(8), 1279 - 83
Conversion of a cyanhydrin compound into S-(-)-3-phenyllactic acid by enantioselective hydrolytic activity of Pseudomonas sp . BC-18; Hashimoto Y et al.; A Pseudomonas strain, named BC-18, which can convert racemic phenylacetaldehyde-cyanhydrin (3-phenyllactonitrile) enantioselectively to S-(-)-3-phenyllactic acid (S-PLA), was isolated from soil . Although PLA produced with intact cells contained the S enantiomers of approximately 75% enantiomeric excess (% e.e.), repeated crystallization gave a higher purity (99.8% e.e.) of the S configuration product . Production of S-PLA was significantly increased when 2.0% (w/v) of calcium chloride were added to the reaction mixture for precipitation of S-PLA . Chemical mutagenesis yielded a mutant strain, named BC348-9, with 16 times higher activity (40 mU/OD630), compared with that of the parent strain (2.5 mU/OD630) . When the mutant strain BC348-9 was used, approximately 18 g/OD630 was produced, which is 12 times higher than that of the parent strain . The final accumulation of PLA exceeded 6.0%, 1.2 times higher than that of the parent strain.

Chirurg, 1996 Aug, 67(8), 843 - 9
{The superficial femoral vein in aorto-iliac position--suitable as autogenous vascular replacement in deep prosthesis infection?}; Franke S et al.; The current standard therapy of aortic graft infection is connected with a high incidence of operative and late complications . The usefulness of autogenous tissue revascularization techniques as promising therapeutic alternatives is limited by the complexity of these procedures and by the lack of suitable donor vessels . Since several authors have shown that serious venous stasis of the donor leg does not occur after superficial femoral vein (SFV) resection, we have started to use this deep leg vein for arterial in situ reconstructions in the treatment of aortic graft infection . During the past 3 years seven patients have received autogenous SFV grafts in this way . There were no intraoperative deaths . Two critically ill patients with severe Pseudomonas sepsis and extensive retroperitoneal abscess each died on the 4th postoperative day, due to acute massive bleeding in the operating area . In all of the five survivors infection could be eradicated; only one limb had to be amputated . Mild to moderate transient swelling of the lower extremity without stasis was regularly seen after SFV resection . During follow-up one patient died of cardiac arrest 5 months postoperatively . The remaining four patients are still alive with patent SFV grafts at 6, 22, 30 and 36 months after the operation.

Diagn Cytopathol, 1996 Aug, 15(2), 98 - 102
Chronic granulomatous disease of childhood: respiratory cytology; Abati A et al.; Chronic granulomatous disease (CGD) of childhood is a rare inherited disease in which phagocytic cells fail to produce the normal respiratory burst in response to infectious stimuli, leaving the patient particularly susceptible to infections with bacteria and fungi that produce catalase . Between 1988 and 1993 at the NIH, 58 pulmonary cytology specimens were obtained on 24 CGD patients . The number of specimens per patient ranged from one to 13 with an average 2.4 . The 58 specimens included: 33 bronchoalveolar lavages; one bronchial brushing; 20 lung or pleural mass fine-needle aspirates; three pleural fluids, and one sputum . Two lung aspirates with insufficient material, five bronchoalveolar lavages performed post-treatment to confirm clinical resolution of disease, and two bronchoalveolar lavages not submitted for culture were excluded from further analysis . Of the 49 remaining specimens obtained from patients clinically suspected of having a pulmonary infection, cytology demonstrated a pathogenic organism in nine (18%) . Microbiologic cultures were positive in 19/49 (39%) . Cytology identified fungus in 8/13 (62%) of documented fungal infections, including four cases where microbiology was negative . Bacterial and mixed bacterial/fungal infections were usually not detected by cytology even with appropriate strains . No organisms were identified by cytology in the four cases of nocardia or the three cases of pseudomonas infection . The combination of cytology and microbiology provided the greatest diagnostic sensitivity, yielding a diagnosis in 22/49 cases (45%) . Of the 27 cases with negative cytology and microbiology, an infectious agent was identified in eight upon submission of additional material: three cytology specimens and five tissue specimens . In the remaining 19 cases, no organisms were identified, however, the patients were treated presumptively . Characteristic pathologic features of granulomatous inflammation, necrosis, and giant cells were present in fine-needle aspirates, often when on organisms could be identified, but were not seen in other types of respiratory specimens.

Zentralbl Veterinarmed B, 1996 Aug, 43(6), 371 - 8
Experimental melioidosis in hens; Vesselinova A et al.; Experimental intramuscular infection of hens with Pseudomonas pseudomallei, strain 2796 (1 x 10(9) CFU from a 24-h culture) was reproduced . Clinical, paraclinical and pathomorphological findings were followed from 1 to 30 days after challenge . Haemagglutinin titre, bacterial dissemination in the viscera, number of leucocytes, alveolar (aMa) and peritoneal (pMa) macrophages and their phagocytic activity in vitro were studied . During the course of infection a leucocytosis as well as an increased haemagglutinin titre (1:256) were established . The number of bacteria per gram tissue in the spleen and liver was highest at 1 day post-infection (p.i.) . Melioidose bacteria from egg yolk were isolated at 15 and 30 days p.i . Leucocyte and pMa phagocytic activity was maximal at 3 days p.i . unlike the activity of aMa which increased gradually until the end of the study . Inflammatory-necrotic changes were found in the viscera and brain at 3 and 15 days p.i . The investigation of experimental melioidosis infection in hens showed that they are susceptible to P . pseudomallei and this disease takes a generalized subacute course.

Plant Cell, 1996 Aug, 8(8), 1225 - 37
Systemic resistance in Arabidopsis induced by biocontrol bacteria is independent of salicylic acid accumulation and pathogenesis-related gene expression; Pieterse CM et al.; Systemic acquired resistance is a pathogen-inducible defense mechanism in plants . The resistant state is dependent on endogenous accumulation of salicylic acid (SA) and is characterized by the activation of genes encoding pathogenesis-related (PR) proteins . Recently, selected nonpathogenic, root-colonizing biocontrol bacteria have been shown to trigger a systemic resistance response as well . To study the molecular basis underlying this type of systemic resistance, we developed an Arabidopsis-based model system using Fusarium oxysporum f sp raphani and Pseudomonas syringae pv tomato as challenging pathogens . Colonization of the rhizosphere by the biological control strain WCS417r of P . fluorescens resulted in a plant-mediated resistance response that significantly reduced symptoms elicited by both challenging pathogens . Moreover, growth of P . syringae in infected leaves was strongly inhibited in P . fluorescens WCS417r-treated plants . Transgenic Arabidopsis NahG plants, unable to accumulate SA, and wild-type plants were equally responsive to P . fluorescens WCS417r-mediated induction of resistance . Furthermore, P . fluorescens WCS417r-mediated systemic resistance did not coincide with the accumulation of PR mRNAs before challenge inoculation . These results indicate that P . fluorescens WCS417r induces a pathway different from the one that controls classic systemic acquired resistance and that this pathway leads to a form of systemic resistance independent of SA accumulation and PR gene expression.

Biochemistry, 1996 Jul 16, 35(28), 9106 - 19
Recombinant toluene-4-monooxygenase: catalytic and Mössbauer studies of the purified diiron and rieske components of a four-protein complex; Pikus JD et al.; Expression of the tmoA-F gene cluster from Pseudomonas mendocina KRI in Escherichia coli BL21(DE3) produces a catalytically active form of the toluene-4-monooxygenase (T4MO) complex . Here we report the purification and characterization of four soluble proteins required for the in vitro reconstitution of T4MO catalytic activity . These proteins are a diiron hydroxylase (T4MOH), a Riesketype ferredoxin (T4MOC), an effector protein (T4MOD), and an NADH oxidoreductase (T4MOF) . The T4MOH component is composed of the tmoA, tmoB, and tmoE gene products {quaternary structure (alpha beta epsilon)2, Mr approximately 220 kDa} . The T4MOA polypeptide contains two copies of the amino acid sequence motif (D/E)X(28-37)DEXRH; the same motif provides all of the protein-derived ligands to the diiron centers of ribonucleotide reductase, the soluble methane monooxygenase, and the stearoyl-ACP delta 9 desaturase . Mossbauer, optical, and EPR measurements show that the T4MOH contains diiron centers and suggest that the diiron center contains hydroxo bridge(s) in the diferric state, as observed for methane monooxygenase . Mossbauer and EPR measurements also show that the T4MOC contains a Rieske-type iron-sulfur center . This assignment is in accord with the presence of the amino acid sequence motif CPHX(15-17)CX2H, which has also been found in the bacterial, chloroplastic, and mitochondrial Rieske proteins as well as the bacterial NADH-dependent cis-dihydrodiol-forming aromatic dioxygenases . While single-turnover catalytic studies confirm the function of the T4MOH as the hydroxylase, the NADH-dependent multiple-turnover hydroxylation activity is increased by more than 100-fold in the presence of the T4MOC, which mediates highly specific electron transfer between the T4MOF and the T4MOH . The T4MOD can be purified as an 11.6 kDa monomeric protein devoid of cofactors or redox-active metal ions; this component is also detected as a substoichiometric consitutent of the purified T4MOH . The rate of the hydroxylation reaction can be mildly stimulated by the further addition of separately purified T4MOD to the T4MOH, implying the formation of a high affinity, catalytically competent complex between these two components . These characterizations define a novel, four-component oxygenase combining elements from the soluble methane oxidation complex of the methanotrophic bacteria and the aromatic hydroxylation complexes of the soil pseudomonads.

Biochem Biophys Res Commun, 1996 Jul 16, 224(2), 375 - 81
Plasmid-encoded degradation of p-nitrophenol by Pseudomonas cepacia; Prakash D et al.; A Pseudomonas cepacia strain RKJ 200 capable of utilising p-nitrophenol (PNP+) as the sole source of carbon, nitrogen, and energy was isolated by selective enrichment . The degradation of PNP by this strain proceeds through an oxidative route as indicated by the accumulation of nitrite molecules in the culture medium . Initial studies indicate that the degradation of PNP occurs via hydroquinone as shown by thin layer chromatography and gas chromatography studies; hydroquinone is further degraded via the beta-ketoadipate pathway . A plasmid of approximately 50 kilobase pairs was found to be responsible for carrying genes for PNP degradation in this strain . This was based on the facts that the PNP- mutants lacked the plasmid and that the PNP+ phenotype could conjugally be transferred . In addition, the same plasmid also encoded resistance to inorganic zinc ions.

FEBS Lett, 1996 Jul 15, 390(1), 104 - 8
Site-directed mutagenesis of the formate dehydrogenase active centre: role of the His332-Gln313 pair in enzyme catalysis; Tishkov VI et al.; Gln313 and His332 residues in the active centre of NAD(+)-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from the bacterium Pseudomonas sp . 101 are conserved in all FDHs and are equivalent to the glutamate-histidine pair in active sites of D-specific 2-hydroxyacid dehydrogenases . Two mutants of formate dehydrogenase from Pseudomonas sp . 101, Gln313Glu and His332Phe, have been obtained and characterised . The Gln313Glu mutation shifts the pK of the group controlling formate binding from less than 5.5 in wild-type enzyme to 7.6 thus indicating that Gln313 is essential for the broad pH affinity profile towards substrate . His332Phe mutation leads to a complete loss of enzyme activity . The His332Phe mutant is still able to bind coenzyme but not substrate or analogues . The role of histidine in the active centre of FDH is discussed . The protonation state of His332 is not critical for catalysis but vital for substrate binding . A partial positive charge on the histidine imidazole, required for substrate binding, is provided via tight H-bond to the Gln313 carboxamide.

Cell, 1996 Jul 12, 86(1), 123 - 33
Tomato Prf is a member of the leucine-rich repeat class of plant disease resistance genes and lies embedded within the Pto kinase gene cluster; Salmeron JM et al.; In tomato, resistance to Pseudomonas syringae pv . tomato (Pst) strains expressing the avirulence gene avrPto requires the presence of at least two host genes, designated Pto and Prf . Here we report that Prf encodes a protein with leucine-zipper, nucleotide-binding, and leucine-rich repeat motifs, as are found in a number of resistance gene products from other plants . prf mutant alleles (4) were found to carry alterations within the Prf coding sequence . A genomic fragment containing Prf complemented a prf mutant tomato line both for resistance to Pst strains expressing avrPto and for sensitivity to the insecticide Fenthion . Prf resides in the middle of the Pto gene cluster, 24 kb from the Pto gene and 500 bp from the Fen gene.

Cell Immunol, 1996 Jul 10, 171(1), 80 - 6
An improved circularly permuted interleukin 4-toxin is highly cytotoxic to human renal cell carcinoma cells . Introduction of gamma c chain in RCC cells does not improve sensitivity; Puri RK et al.; We have previously demonstrated that a chimeric protein composed of human IL-4 and Pseudomonas exotoxin, termed IL4-PE4E, is cytotoxic to primary cells derived from human renal cell carcinoma (RCC) . To improve the cytotoxicity of IL4-toxins such as IL4-PE4E and IL4-PE38KDEL to IL-4 receptor (IL-4R) positive tumor cells, a circularly permuted chimeric toxin was prepared by fusing a truncated PE gene encoding PE38KDEL 3' to a circularly permuted IL-4 mutant gene encoding IL4 amino acids 38-129, the linker GGNGG, and IL4 amino acids 1-37 . The resulting chimeric protein, termed IL4(38-37)-PE38KDEL, was tested on five RCC cell lines and its cytotoxicity was compared to that of the native IL4-toxins IL4-PE4E and IL4-PE38KDEL . IL4(38-37)-PE38KDEL was found to be 5 to 10 times more cytotoxic to all cell cultures tested compared to either native IL4-toxin . The cytotoxic activity of IL4(38-37)-PE38KDEL was competible by excess IL-4 and was confirmed by clonogenic assay . IL4(38-37)-PE38KDEL bound to IL-4R on RCC cells with 6- to 12-fold higher affinity than IL4-PE38KDEL or IL4-PE4E . RCC tumor cells were found to lack the common gamma chain (gamma c) of the IL-4R reported to be present on immune cells . The stable transfection of RCC cells with the gamma c chain gene did not significantly change their sensitivity to IL4(38-37)-PE38KDEL . Taken together, our results indicate that the CPIL4-toxin IL4(38-37)-PE38KDEL is highly cytotoxic to human RCC cells due to increased binding affinity to IL-4R while it is not cytotoxic or slightly cytotoxic to T and B cells, monocytic cell lines, and fresh resting or activated bone marrow-derived cells . The gamma c does not seem to increase the internalization rate and/or processing of IL4-toxins in RCC cells . CPIL4-toxin may be a useful agent for the treatment of human RCC.

Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 6902 - 6
Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: implications for the activation process and for ADP ribosylation; Li M et al.; The catalytic, or third domain of Pseudomonas exotoxin A (PEIII) catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor-2 in eukaryotic cells, inhibiting protein synthesis . We have determined the structure of PEIII crystallized in the presence of NAD to define the site of binding and mechanism of activation . However, NAD undergoes a slow hydrolysis and the crystal structure revealed only the hydrolysis products, AMP and nicotinamide, bound to the enzyme . To better define the site of NAD binding, we have now crystallized PEIII in the presence of a less hydrolyzable NAD analog, beta-methylene-thiazole-4-carboxamide adenine dinucleotide (beta-TAD), and refined the complex structure at 2.3 angstroms resolution . There are two independent molecules of PEIII in the crystal, and the conformations of beta-TAD show some differences in the two binding sites . The beta-TAD attached to molecule 2 appears to have been hydrolyzed between the pyrophosphate and the nicotinamide ribose . However molecule 1 binds to an intact beta-TAD and has no crystal packing contacts in the vicinity of the binding site, so that the observed conformation and interaction with the PEIII most likely resembles that of NAD bound to PEIII in solution . We have compared this complex with the catalytic domains of diphtheria toxin, heat labile enterotoxin, and pertussis toxin, all three of which it closely resembles.

Int J Cancer, 1996 Jul 3, 67(1), 113 - 23
Recombinant single-chain and disulfide-stabilized Fv-immunotoxins that cause complete regression of a human colon cancer xenograft in nude mice; Reiter Y et al.; Monoclonal antibody (MAb) 55.1 specifically recognizes an antigen on the surface of human colon adenocarcinoma cells . We constructed recombinant immunotoxins composed of the heavy- and light-chain variable regions of MAb 55.1 fused to a recombinant form of Pseudomonas exotoxin (PE) . The heavy- and light-chain variable regions are stabilized by 2 means . One is by a flexible peptide linker to form a single-chain antigen binding protein (scFv) and the second by an interchain disulfide bond engineered between structurally conserved framework regions . These are termed disulfide stabilized Fvs (dsFv) . The 2 Fv forms are fused to truncated forms of PE lacking the cell binding domain . The recombinant scFv- and dsFv-immunotoxins were expressed in E . coli and purified to near homogeneity . The scFv- and dsFv-immunotoxins were shown to be specifically cytotoxic to human colon adenocarcinoma cell lines . The scFv-immunotoxin containing PE38KDEL was more active than the immunotoxin containing PE38 with the native carboxyl terminus (REDLK) . However, the PE38KDEL immunotoxin is about 2-fold more toxic in mice, and therefore it does not appreciably increase the therapeutic window in mice . Intravenous administration of the scFv- and dsFv- recombinant immunotoxins caused complete regression of a human colon carcinoma (Colo205) growing subcutaneously in immunodeficient mice . The dsFv-immunotoxin has better antitumor activity compared with its scFv-immunotoxin counterpart.

Zh Mikrobiol Epidemiol Immunobiol, 1996 Jul-Aug, (4), 75 - 8
{The immunogenicity and heterogeneity of Pseudomonas pseudomallei surface antigen 8}; Piven' NN et al.; Surface antigen 8, one of pathogenicity factors of P.pseudomallei and having an antiphagocytic action, contains glycoprotein with a mol . wt . of 200 kD and proteins with mol wt . of 25, 30 and 34 kD . According to its chemical structure, the carbohydrate part of antigen 8 is the homogeneous polymer of 6-d-D-mannoheptose, and its protein component is a collection of monomer proteins with mol . wt . of 12-120 kD . The consecutive fractionation of antigen 8 by gel and ion exchange chromatography has made it possible to isolate its individual fragments, differing by the collection of antigenic components, as well as by their antiphagocytic and protective activity . Experiments have shown that glycoprotein with a mol . wt . of 200 kD is an active immunosuppressive agent, while protein with a mol . wt . of 34 kD produces a pronounced immunogenic effect.

Biochem Mol Biol Int, 1996 Jul, 39(4), 781 - 8
Amino acid sequence of catechol 1,2-dioxygenase (pyrocatechase) isozyme alpha alpha from Pseudomonas arvilla C-1; Nakai C et al.; Catechol 1,2-dioxygenase catalyzes the oxygenative ring cleavage of catechol to form cis,cis-muconic acid, and contains a ferric form of iron as its sole cofactor . Pseudomonas arvilla C-1 contains three isozymes of the enzyme, alpha alpha, alpha beta, and beta beta {C . Nakai et al . (1990) J . Biol . Chem . 265, 660-665} . We have determined the amino acid sequence of the alpha alpha isozyme by direct analysis . The sequence shared 77% homology with isozyme beta beta, and had conserved tyrosyl and histidyl residues which are thought to be involved in the binding of ferric ion.

Radiographics, 1996 Jul, 16(4), 855 - 69
Imaging of pulmonary-cutaneous disorders: matching the radiologic and dermatologic findings; Franquet T et al.; Dermatologic lesions are often associated with pulmonary disorders and vice versa . Diseases with pulmonary and cutaneous manifestations can be divided into four major categories: (a) congenital and developmental disorders with cutaneous-pulmonary manifestations (Ehlers-Danlos syndrome, generalized elastolysis, yellow nail syndrome, neurofibromatosis, hereditary hemorrhagic telangiectasia); (b) primary dermal diseases with associated pulmonary manifestations (septic vasculitis, malignant melanoma, Kaposi sarcoma); (c) primary pulmonary diseases with associated cutaneous manifestations (tuberculosis, Pseudomonas pneumonia, mycoplasmal pneumonia, adenocarcinoma, metastasis); and (d) cutaneous-pulmonary conditions (multisystem disorders) (progressive systemic sclerosis, systemic lupus erythematosus, Wegener granulomatosis, sarcoidosis) . A series of selected cases is used to illustrate the radiologic and dermatologic features of conditions that affect both the lung and dermal tissue . Specific emphasis is placed on the dermatologic manifestations of disease . Diagnosis of a pulmonary-cutaneous disorder requires familiarity with the morphologic appearance of the cutaneous lesion.

Ann Pharmacother, 1996 Jul-Aug, 30(7-8), 840 - 50
Inhaled antibiotics in cystic fibrosis: a review; Toso C et al.; OBJECTIVE: To provide an overview of aerosol drug therapy, including physical considerations and aerosol drug delivery systems, and to review clinical experience with inhaled antibiotics in cystic fibrosis (CF) when used as adjunctive therapy to intravenous therapy for acute pulmonary exacerbations and chronic, suppressive therapy . DATA SOURCES: A MEDLINE search (1966-1995) of English-language literature describing the use of inhaled antibiotics for the management of CF . STUDY SELECTION AND DATA EXTRACTION: All articles were considered for possible inclusion in the review . Pertinent information as judged by the authors was selected for discussion . DATA SYNTHESIS: The use of inhaled antibiotics as adjunctive therapy to systemic therapy for acute exacerbations did not improve pulmonary function tests, increase hospital discharge rate, or permanently eradicate sputum Pseudomonas . Clinical trials of inhaled antibiotics as suppressive therapy yielded variable results . Individually, four trials documented a significant improvement in pulmonary function, three trials documented a slower decline in pulmonary function, and four trials reported a reduced frequency of hospitalizations . However, the trials were unable to collectively document a prolonged beneficial effect of inhaled antibiotics on pulmonary function, sputum bacterial density, and frequency of hospitalizations . CONCLUSIONS: Clinical trials conducted thus far suggest no role for inhaled antibiotics in the treatment of acute pulmonary exacerbations in patients with CF . Aerosolized antibiotics used as suppressive therapy may be useful in certain patients, but their use should be limited to select patients based on individual response to therapy . Additional long-term, well-controlled trials of inhaled antibiotics as suppressive therapy are needed before routine use can be recommended.

Poult Sci, 1996 Jul, 75(7), 854 - 6
Relevance of water quality to broiler and turkey performance; Barton TL; Water was tested from 300 broiler farms in Arkansas in cooperation with three integrated poultry companies, each having at least two locations in the state . The turkey study was conducted in cooperation with three integrated turkey companies with samples from 100 turkey farms, although the numbers were not equal among companies . Performance criteria collected were body weight, feed conversion, livability, and condemnation . In the overall analysis in the broiler study, nitrate had a detrimental effect on performance . Calcium was negatively correlated with adjusted conversion; i.e., conversion improved as calcium increased . Magnesium was positively correlated with adjusted conversion, or had an adverse effect on conversion . Dissolved oxygen, bicarbonate, hardness, calcium, and magnesium were all positively correlated with adjusted weight but nitrate was negatively correlated with adjusted weight . Calcium and potassium were negatively correlated with livability and calcium and nitrate were positively correlated with condemnation . The bacterial results showed no significant difference between top and bottom producers for either Pseudomonas or Escherichia coli . In the turkey study, calcium, magnesium, bicarbonate, hardness, and aggressive index were beneficial to feed conversion . Phosphate and ammonia were detrimental to feed conversion . Calcium, magnesium, dissolved oxygen, zinc, hardness, and aggressive index were all positively correlated with adjusted body weight . Magnesium was negatively correlated with livability . Magnesium and aggressive index were positively correlated with condemnation and potassium, zinc, nitrate, and phosphate were negatively correlated with condemnation . Although fewer farms were involved in the turkey study, the results generally support the results of the broiler study.

Pancreas, 1996 Jul, 13(1), 16 - 21
Cytotoxic effects of TGF-alpha-Pseudomonas exotoxin A fusion protein in human pancreatic carcinoma cells; Baldwin RL et al.; The epidermal growth factor (EGF) receptor is overexpressed in human pancreatic cancers and cultured cell lines . TP40 is a chimeric protein composed of transforming growth factor-alpha (TGF-alpha) linked to a modified Pseudomonas exotoxin A (PE40) that exerts growth inhibitory effects on cells bearing a high number of EGF receptors . Therefore, we compared the effect of TP40 on the growth of Chinese hamster ovary (CHO), cells expressing varying levels of the EGF receptor and on the growth of two human pancreatic cancer cell lines . The growth of CHO cells devoid of endogenous EGF receptors was minimally altered by high concentrations of TP40, even following a 72-h incubation period . In contrast, in CHO cells expressing approximately 95,000 and 438,000 EGF receptors per cell, one-half maximal growth inhibition occurred at 5 and 3 ng/ml TP40, respectively . Following a 72-h incubation in T3M4 and COLO 357 human pancreatic cancer cells, one-half maximal growth inhibition occurred at 0.2 and 0.4 ng/ml TP40, respectively . This effect was significantly greater than that of native Pseudomonas exotoxin A . These findings indicate that human pancreatic cancer cells are markedly sensitive to the growth inhibitory effects of TP40 and raise the possibility that TP40 may have a therapeutic role in this disorder.

Int J Biol Macromol, 1996 Jul, 19(1), 29 - 34
Sequential production of two different polyesters in the inclusion bodies of Pseudomonas oleovorans; Curley JM et al.; When Pseudomonas oleovorans was grown on a mixture of 5-phenylvaleric acid, PVA, and nonanoic acid, NA, the reserve polyester produced included both a homopolymer and a copolymer . The homopolymer poly-3-hydroxy-5-phenylvalerate, PHPV, contained only 3-hydroxy-5-phenylvalerate units, while the copolymer contained the same long chain 3-hydroxyalkanoates as those present in the copolymer poly-3-hydroxynonanoate, PHN, which is produced from acid alone . The intracellular location of each of these polymers was determined by selective staining of the inclusion body granules with ruthenium tetraoxide and examination by transmission electron microscopy showed that both types of polyesters occurred in the same granule . PHN was present in the center of the granule, while PHPV accumulated around the PHN in the inclusion body . The proteins associated with the inclusion bodies were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) . In all cases, two different polymerase enzymes of molecular weight 59 and 55 KDa were present, indicating that the same polymerase enzyme system was responsible for the production of both PHN and PHPV . Attempts were made to produce a random copolymer containing both alkyl and phenylalkyl repeat units by varying the growth conditions, but a mixture of PHN and PHPV was always produced instead.

Int J Syst Bacteriol, 1996 Jul, 46(3), 802 - 10
Emended description of Herbaspirillum; inclusion of {Pseudomonas} rubrisubalbicans, a milk plant pathogen, as Herbaspirillum rubrisubalbicans comb . nov.; and classification of a group of clinical isolates (EF group 1) as Herbaspirillum species 3; Baldani JI et al.; {Pseudomonas} rubrisubalbicans, a mild plant pathogen . Herbaspirillum seropedicae, and EF group 1 strains (clustered by an immunological method) were investigated by a polyphasic approach with DNA-rRNA and DNA-DNA hybridizations and auxanography on 147 substrates . Our results show that they all belong to the genus Herbaspirillum . In addition to H . seropedicae, two other species are described: Herbaspirillum rubrisubalbicans and a new unnamed species, Herbaspirillum species 3, containing mainly strains of clinical origin . The three species can be differentiated on the basis of their auxanographic features and DNA-DNA similarities . The type strain of H . rubrisubalbicans is NCPPB 1027 (=LMG 2286); representative strains of the third Herbaspirillum species are strains CCUG 189 (=LMG 5523), CCUG 10263 (=LMG 5934), and CCUG 11060 (=LMG 5321) . It has been confirmed that H . rubrisubalbicans is an endophytic diazotroph . It colonizes the roots, the stems, and predominantly the leaves of sugarcane (Saccharum spp.), while Herbaspirillum seropedicae colonizes in large numbers many different species of the Gramineae . Both diazotrophic Herbaspirillum species could be differentiated with meso-erythritol and N-acetylglucosamine . Oligonucleotide probes based on partial sequences of the 23S rRNA of H . seropedicae and H . rubrisubalbicans (HS and HR probes, respectively), were constructed and used as diagnostic probes.

Int J Syst Bacteriol, 1996 Jul, 46(3), 769 - 73
Isolation and identification of poly(3-hydroxyvalerate)-degrading strains of Pseudomonas lemoignei; Mergaert J et al.; By using selective enrichment of polyhydroxyalkanoate-degrading bacteria and poly(3-hydroxyvalerate)-containing granules from Chromobacterium violaceum as the carbon source, 10 new Pseudomonas lemoignei strains were isolated; these strains were able to degrade poly(3-hydroxyvalerate), as well as poly(3-hydroxybutyrate), in vitro . The new isolates were characterized and identified by comparing them with P . lemoignei LMG 2207(T) (T = type strain) . Like P . lemoignei LMG 2207(T) cells, the cells of the 10 new isolates contained mainly hexadecenoic, hexadecanoic, octadecenoic, and dodecanoic acids, as well as hydroxylated fatty acids, and exhibited respiration in the presence of methylpyruvate, 3-hydroxybutyrate, and 4-hydroxybutyrate, but not in the presence of the 92 other carbon sources included in Biolog GN microplates . The protein patterns of the new isolates were almost identical to each other and very similar to the protein pattern of P . lemoignei LMG 2207(T) . Some of the new isolates, but not P . lemoignei LMG 2207(T), contained megaplasmids that were about 200 kbp long . The 16S ribosomal DNA genes of strain A62, a representative of the 10 new isolates, and of P . lemoignei LMG 2207(T) exhibited more than 0.99 sequence similarity . The DNA-DNA reassociation value for two representative strains was 100%, and the levels of DNA-DNA reassociation between these strains and the type strain were 60 and 61% . The taxonomy of P . lemoignei is briefly discussed.

Biosci Biotechnol Biochem, 1996 Jul, 60(7), 1056 - 62
Cloning and characterization of the gene encoding pyrroloquinoline quinone-dependent poly(vinyl alcohol) dehydrogenase of Pseudomonas sp . strain VM15C; Shimao M et al.; A gene library of poly(vinyl alcohol) (PVA)-degrading Pseudomonas sp . strain VM15C was constructed in Escherichia coli with the vector pUC18 . Screening of this library with a chromogenic PVA dehydrogenase assay resulted in the isolation of a clone that carries the gene (pdh) for the PVA dehydrogenase, and the entire nucleotide sequence of its structural gene was determined . The gene encodes a protein of 639 amino acid residues (68,045 Da) and in the deduced amino acid sequence, some putative functional sites, a signal sequence, a heme c-binding site, and a PQQ-binding site, were detected . The amino acid sequence showed low similarity to other types of quinoprotein dehydrogenases . PVA dehydrogenase expressed in E . coli clones required PQQ . Ca2+, and Mg2+ stimulated the activity . PVA-dependent heme c reduction occurred with exogenous PQQ in cell extracts of the E . coli clone . The PVA dehydrogenase in the E . coli clone was localized in the cytoplasm.

Appl Environ Microbiol, 1996 Jul, 62(7), 2360 - 74
Distribution of Pseudomonas syringae pathovars into twenty-three O serogroups; Saunier M et al.; Serological reactions of Pseudomonas syringae and Pseudomonas viridiflava were studied by Ouchterlony double diffusion . A total of 55 polyclonal antisera, containing anti-lipopolysaccharide (anti-LPS) precipitating antibodies, were cross-tested against antigenic suspensions of 51 strains . Twenty-three O serogroups were defined, primarily on the reaction of the type strains . Two families of O serogroups showed antigenic crossreactivities (PHA, MOP1, MOP2, MOP3, HEL1, HEL2, and SYR1; PERSAVTOM1, PERSAVTOM2, DEL, POR, and SYR2) . Ten O serogroups showed a clearcut specificity: APTPIS, TAB, VIR1, VIR2, VIR3, SYR3, SYR4, SYR5, HUS, and LAC . The last serogroup (RIB) contained strains with rough colony morphology and side chain-deficient LPSs, as evidenced by sodium dodecyl sulfate-polycrylamide gel electrophoresis . The LPS basis of the O serogroups was demonstrated by immunoblotting . Serological reference strains were designated for all of the O serogroups and correspondence was established between the O serogroups studied and seven previous serogroups (L . T . Pastushenko and I.D . Simonovich, Mikrobiol, Zh . 41:222-229 and 330-339, 1979) . A total of 355 strains of P . syringae (sensu lato) belonging to 15 pathovars, not including pathovar syringae, were typed into the 23 described O serogroups . O serogroups were assigned after double-diffusion reactions, with each strain compared with serological references . The utility of O serogrouping to study P . syringae pathovar structure and diversity is discussed.

J Bacteriol, 1996 Jul, 178(14), 4027 - 30
Alkylresorcinols are abundant lipid components in different strains of Azotobacter chroococcum and Pseudomonas spp; Kozubek A et al.; The occurrence of various amounts of 5-n-alkylresorcinols was shown in lipids extracted from 14 bacterial strains of Azotobacter chroococcum as well as from strains of Pseudomonas aureofaciens, P . chlororapsis, and P . fluorescens . The amount of alkylresorcinols found varied from 2.3 to 56.2 microg/mg (dry weight) of cells in A . chroococum and from 0.2 to 0.8 microg/mg (dry weight) of cells in Pseudomonas spp . Strains of both genera produce saturated homologs with C13 to C27 side chains . C19, C21, and C23 homologs are predominant in and characteristic for A . chroococum strains, the C15 homolog is predominant in and characteristic for P . chlororapsis and P . fluorescens, and the C17 homolog is predominant in and characteristic for P . aureofaciens . The presence of 5-n-(2-ketoalkyl)resorcinols, not previously observed, was demonstrated in lipids isolated from the cells of A . chroococum Az5.

Plant J, 1996 Jul, 10(1), 71 - 82
Benzothiadiazole induces disease resistance in Arabidopsis by activation of the systemic acquired resistance signal transduction pathway; Lawton KA et al.; Benzothiadiazole (BTH) is a novel chemical activator of disease resistance in tobacco, wheat and other important agricultural plants . In this report, it is shown that BTH works by activating SAR in Arabidopsis thaliana . BTH-treated plants were resistant to infection by turnip crinkle virus, Pseudomonas syringae pv 'tomato' DC3000 and Peronospora parasitica . Chemical treatment induced accumulation of mRNAs from the SAR-associated genes, PR-1, PR-2 and PR-5 . BTH treatment induced both PR-1 mRNA accumulation and resistance against P . parasitica in the ethylene response mutants, etr1 and ein2, and in the methyl jasmonate-insensitive mutant, jar1, suggesting that BTH action is independent of these plant hormones . BTH treatment also induced both PR-1 mRNA accumulation and P . parasitica resistance in transgenic Arabidopsis plants expressing the nahG gene, suggesting that BTH action does not require salicylic acid accumulation . However, because BTH-treatment failed to induce either PR-1 mRNA accumulation or P . parasitica resistance in the non-inducible immunity mutant, nim1, it appears that BTH activates the SAR signal transduction pathway.

J Bacteriol, 1996 Jul, 178(13), 3727 - 35
Growth phase-dependent transcription of the sigma(54)-dependent Po promoter controlling the Pseudomonas-derived (methyl)phenol dmp operon of pVI150; Sze CC et al.; Transcription from Pseudomonas-derived -24, -12 Po promoter of the pVI150-encoded dmp operon is mediated by the sigma 54-dependent DmpR activator in response to the presence of aromatic pathway substrates in the medium . However, global regulatory mechanisms are superimposed on this regulatory system so that the specific response to aromatic effectors is absent in cultures until the stationary phase is reached . Here we genetically dissect the system to show that the growth phase response is faithfully mimicked by a minimal system composed of the dmpR regulatory gene and the Po promoter regulatory region and can be reproduced in heterologous Escherichia coli . Using this system, we show that the growth phase-dependent DmpR-mediated response to aromatic compounds is limited to fast-growing cultures . Thus, during exponential growth of cultures in minimal media containing different carbon sources, the response to aromatics is immediate, while the response is suppressed in cultures grown on rich media until the exponential-to-stationary phase transition . Elements known to be involved in the DmpR-mediated transcription from Po were analyzed for the ability to influence the growth phase response . Most dramatically, overexpression of DmpR was shown to completely abolish the growth phase response, suggesting that a negatively acting factor may mediate this level of regulation . The possible mechanism of action and integration (of the specific regulation of the dmp operon-encoded catabolic enzymes with the physiological status of the bacteria are discussed.

Curr Microbiol, 1996 Jul, 33(1), 16 - 25
Effect of Different Temperature Upshifts on Protein Synthesis by the Psychrotrophic Bacterium Pseudomonas fragi
Michel V V, Labadie J, Hebraud M.
Pseudomonas fragi, a psychrotroph bacterium involved in meat product spoilage, was shifted either from 5degrees to 20degreesC or 30degreesC and from 28degrees to 34degreesC . The heat-shocked cells in the mid-log phase rapidly reached the characteristic growth rate of the postshock temperature . The patterns of synthesized proteins were compared by autoradiography of two-dimensional gel electrophoregrams . The rates of synthesis, after transfer of cells from 5degrees to 30degreesC, 5degrees to 20degreesC, and 28degrees to 34degreesC, changed for 30, 26, and 21 proteins respectively, of which 19, 17, and 12 were increased respectively . Thirteen proteins changed similarly for the three treatments, and two of the seven overexpressed proteins were immunologically related to the Escherichia coli DnaK and GroEL heat shock proteins . From the four low-molecular-mass proteins, belonging to the family of DNA-binding cold shock proteins (CSPs) such as CS7.4, the major E . coli CSP {15}, the amounts of C7.0 and C8.0 decreased rapidly after the upshifts, whereas that of E7.0 and E8.0 increased greatly.

Microb Ecol, 1996 Jul, 32(1), 23 - 33
Exopolysaccharide Production and Attachment Strength of Bacteria and Diatoms on Substrates with Different Surface Tensions
Becker K.
Attachment strength and exopolysaccharide (EPS) production of Pseudomonas sp . (bacteria) and the diatom Amphora coffaeformis were studied on six different substrata with surface tensions between 19 and 64.5 mN m-1 . Test panels of the materials were exposed to bacterial cultures between 3 and 120 hours, and to diatom cultures between 48 and 72 hours . Exopolysaccharide production by surface-associated cells was measured using the phenol sulfuric acid method . Attachment studies were run by exposing test panels to laminar flow pressure using a radial flow chamber . Highest EPS production by bacteria and diatoms was recorded on substrata with surface tensions above 30 mN m-1 . Lowest EPS production occurred on substrata between 20 and 25 mN m-1 . Highest EPS production and strongest adhesion was found on polycarbonate (33.5 mN m-1) . Both test organisms improved their attachment strength with exposure time on most materials . However, amounts of produced EPS and improvement of attachment indicated that mechanisms other than polysaccharide production are more important on substrata with low surface tensions (<25 mN m-1) . Simply producing more polysaccharides is not sufficient to overcome weak attachment on materials with low surface tensions . For example, adhesion of Pseudomonas sp . and A . coffaeformis on polytetrafluorethylene/perfluor-copolymer (PFA; 22 mN m-1) and glass (64.5 mN m-1) was equally strong although EPS production was much higher on glass than on PFA . This is somewhat surprising for A . coffaeformis because polysaccharide production has been considered the most important attachment mechanism of A . coffaeformis.

Biochemistry, 1996 Jun 25, 35(25), 8103 - 9
Structure of 4-chlorobenzoyl coenzyme A dehalogenase determined to 1.8 A resolution: an enzyme catalyst generated via adaptive mutation; Benning MM et al.; Here we describe the three-dimensional structure of 4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp . strain CBS-3 . This enzyme catalyzes the hydrolysis of 4-chlorobenzoyl-CoA to 4-hydroxybenzoyl-CoA . The molecular structure of the enzyme/4-hydroxybenzoyl-CoA complex was solved by the techniques of multiple isomorphous replacement, solvent flattening, and molecular averaging . Least-squares refinement of the protein model reduced the crystallographic R factor to 18.8% for all measured X-ray data from 30 to 1.8 A resolution . The crystallographic investigation of this dehalogenase revealed that the enzyme is a trimer . Each subunit of the trimer folds into two distinct motifs . The larger, N-terminal domain is characterized by 10 strands of beta-pleated sheet that form two distinct layers which lie nearly perpendicular to one another . These layers of beta-sheet are flanked on either side by alpha-helices . The C-terminal domain extends away from the body of the molecule and is composed of three amphiphilic alpha-helices . This smaller domain is primarily involved in trimerization . The two domains of the subunit are linked together by a cation, most likely a calcium ion . The 4-hydroxybenzoyl-CoA molecule adopts a curved conformation within the active site such that the 4-hydroxybenzoyl and the adenosine moieties are buried while the pantothenate and pyrophosphate groups of the coenzyme are more solvent exposed . From the three-dimensional structure it is clear that Asp 145 provides the side-chain carboxylate group that adds to form the Meisenheimer intermediate and His 90 serves as the general base in the subsequent hydrolysis step . Many of the structural principles derived from this investigation may be directly applicable to other related enzymes such as crotonase.

J Mol Biol, 1996 Jun 21, 259(4), 704 - 17
Crystal structure of a bacterial lipase from Chromobacterium viscosum ATCC 6918 refined at 1.6 angstroms resolution; Lang D et al.; The crystal structure of a lipase from the bacterium Chromobacterium viscosum ATCC 6918 (CVL) has been determined by isomorphous replacement and refined at 1.6 angstroms resolution to an R-factor of 17.8% . The lipase has the overall topology of an alpha/beta type protein, which was also found for previously determined lipase structures . The catalytic triad of the active center consists of the residues Ser87, Asp263 and His285 . These residues are not exposed to the solvent, but a narrow channel connects them with the molecular surface . This conformation is very similar to the previously reported closed conformation of Pseudomonas glumae lipase (PGL), but superposition of the two lipase structures reveals several conformational differences . r.m.s . deviations greater than 2 angstroms are found for the C alpha-atoms of the polypeptide chains from His15 to Asp28, from Leu49 to Ser54 and from Lys128 to Gln158 . Compared to the PGL structure in the CVL structure, three alpha-helical fragments are shorter, one beta-strand is longer and an additional antiparallel beta-sheet is found . In contrast to PGL, CVL displays an oxyanion hole, which is stabilized by the amide nitrogen atoms of Leu17 and Gln88, and a cis-peptide bond between Gln291 and Leu292 . CVL contains a Ca2+, like the PGL, which is coordinated by four oxygen atoms from the protein and two water molecules.

Biochemistry, 1996 Jun 18, 35(24), 7834 - 45
Active site structure of Rieske-type proteins: electron nuclear double resonance studies of isotopically labeled phthalate dioxygenase from Pseudomonas cepacia and Rieske protein from Rhodobacter capsulatus and molecular modeling studies of a Rieske center; Gurbiel RJ et al.; Continuous wave electron nuclear double resonance (CW ENDOR) spectra of {delta-15N,epsilon(-14)N}histidine-labeled phthalate dioxygenase (PDO) from Pseudomonas cepacia were recorded and found to be virtually identical to those previously recorded from {delta,epsilon-15N2}histidine-labeled protein {Gurbiel, R . J., Batie, C . J., Sivaraja, M., True, A . E., Fee, J . A., Hoffman, B . M., & Ballou, D . P . (1989) Biochemistry 28, 4861-4871} . Thus, the two histidine residues, previously shown to ligate one of the irons in the cluster {cf . Gurbiel et al . 1989)}, both coordinate the metal at the N(delta) position of their imidazole rings . Pulsed ENDOR studies showed that the "remote", noncoordinating nitrogen of the histidine imidazole ring could be observed from the Rieske protein in a sample of Rhodobacter capsulatus cytochrome bc1 complex uniformly labeled with 15N but not in a sample of PDO labeled with {delta-15N,epsilon-14N}histidine, but this atom was easily observed with a sample of Rh . capsulatus cytochrome bc1 complex that had been uniformly labeled with 15N; this confirmed the conclusion from the CW ENDOR studies that ligation is exclusively via N(delta) for both ligands in the PDO center . Modifications in the algorithms previously used to simulate 14N ENDOR spectra permitted us to compute spectra without any constraints on the relative orientation of hyperfine and quadrupole tensors . This new algorithm was used to analyze current and previously published spectra, and slightly different values for the N-Fe-N angle and imidazole ring rotation angles are presented {cf . Gurbiel et al . (1989) Gurbiel, R . J., Ohnishi, T., Robertson, D . E., Daldal, F., and Hoffman, B . M . (1991) Biochemistry 30, 11579-11584} . This analysis has permitted us to refine the proposed structure of the {2Fe-2S} Rieske-type cluster and rationalize some of the properties of these novel centers . Although the spectra of cytochrome bc1 complex from Rh . capsulatus are of somewhat lower resolution than those obtained with samples of PDO, our analysis nevertheless permits the conclusion that the geometry of the cluster is essentially the same for all Rieske and Rieske-type proteins . Structural constraints inferred from the spectroscopic results permitted us to apply the principles of distance geometry to arrive at possible three-dimensional models of the active site structure of Rieske protein from Rh . capsulatus . Results from this test case indicate that similar procedures should be generally useful in metalloprotein systems . We also recorded the pulsed and CW ENDOR spectra of 57Fe-labeled PDO, and the resulting data were used to derive the full hyperfine tensors for both Fe(III) and Fe(II) ions, including their orientations relative to the g tensor . The A tensor of the ferric ion is nominally isotropic, while the A tensor of the ferrous ion is axial, having A(parallel) > A(perpendicular); both tensors are coincident with the observed g tensor, with A(parallel) of the ferrous ion lying along the maximum g-value, g1 . These results were examined using refinements of existing theories of spin-coupling in {2Fe-2S}+ clusters, and it is concluded that current theories are not adequate to fully describe the experimental results.

FEMS Microbiol Lett, 1996 Jun 15, 140(1), 37 - 42
Characterisation of carbon dioxide-inducible genes of the marine bacterium, Pseudomonas sp . S91; Stretton S et al.; Characterisation of two genes in Pseudomonas sp . S91 that are responsive to carbon dioxide is reported . These were identified by random transposon mutagenesis leading to fusion of the Escherichia coli lacZ reporter gene to the genes of interest . Expression of the genes' promoters was quantified by measuring the reporter gene product, beta-galactosidase . beta-Galactosidase synthesis was induced when cells were exposed to 10% CO2 on solid media or during growth in aqueous phase when the culture density was greater than 1 at 610 nm, in either rich or minimal media . Induction of beta-galactosidase synthesis was not due to: increased alkalinity, onset of stationary phase, build up of soluble metabolites in the culture supernatant, or cell density-dependent signalling . The CO2-inducible gene fusions were not induced by other environmental conditions that are known to stimulate global regulators of environmental gene expression . Benzoic acid (2 mM) induced beta-galactosidase synthesis in one of the mutants indicating the Co2 response may involve the intracellular CO2 partial pressure/bicarbonate ion concentration/pH equilibrium.

Biochim Biophys Acta, 1996 Jun 11, 1281(2), 189 - 204
Assessment of molecular sieving across bacterial outer membrane of Pseudomonas; Kulkarni SB et al.; The role of the permeability barrier of the outer membrane of Pseudomonas was re-evaluated based on the physical theory of molecular sieving in view of its intrinsic antibiotic resistance . We developed a set of analytical procedures based on parametric and non-parametric statistical tests to evaluate, validate and adopt the better among a set of competing non-linear models of diffusion . The molecular mass dependence of uptake of non-electrolytes in bacteria yielded a quantitative measure to distinguish between sieving mechanisms and specific uptake/efflux mechanisms . The experimental data, supported by the physical model of DEAE-Sephadex and various analytical models and extensive simulation of the errors, both in measurement and models, yielded evidence consistent with the relaxation of the outer membrane matrix barrier in Pseudomonas.

Pol Tyg Lek, 1996 Jun, 51(23-26), 331 - 3
{Use of human anti-Pseudomonas immunoglobulins in treatment of children with burns}; Bukowska D et al.; This studies included 15 children with burns involving 10-55% of the whole body surface, treated at the two surgical departments in Poland . All patients have been given 0.5 mL of a 15% solution of anti-Pseudomonas immunoglobulin in a deep i.m . injections for 3 consecutive days . Immunoglobulin has generally been well tolerated, except short fever attacks . Human anti-Pseudomonas immunoglobulin prepared in the institute of Haematology and Transfusion in Warsaw prevented infections with P . aeruginosa in 12 burned children . There have been no cases of bacteremia produced by P . aeruginosa in 15 treated children with burns . The obtained results indicate efficacy of such therapy in burned children.

Immunopharmacology, 1996 Jun, 33(1-3), 369 - 73
Further evidence of bradykinin involvement in septic shock: reduction of kinin production in vivo and improved survival in rats by use of polymer tailored SBTI with longer t1/2; Shin YH et al.; Involvement of bradykinin in septic shock and its therapeutic endeavor using soybean trypsin inhibitor (SBTI, Kunitz type) were investigated in an in vivo model of septic shock induced by pseudomonal elastase . Pseudomonal elastase injection at 0.5 mg/kg i.v . to guinea pigs resulted in elevation level of bradykinin in the blood from < 1 ng/ml to 25 ng/ml which was accompanied by a drop of mean arterial blood pressure (MABP) (about 45 mmHg) . When native soybean trypsin inhibitor (SBTI, Kunitz type, 20 kDa) was injected, into this model, induction of bradykinin generation and hypotension by the bacterial protease treatment was completely obliterated as judged by the both levels of bradykinin and MABP . Specifically, by the treatment with SBTI, bradykinin levels did not increase and the drop of the blood pressure was minimal (< 10 mmHg) in this time frame (< 30 min) . We designed and prepared succinylated gelatin-conjugated SBTI (suc-gel SBTI) with enlarged molecular mass (M(r) approximately 110,000) and higher area under the curve of the plasma concentration, which exhibits about 6 times longer plasma half-life (t1/2) and about 4 times larger area under the curve of plasma concentration . Suc-gel-SBTI suppressed the pseudomonal protease-induced shock much more effectively than native SBTI, the conjugate exhibited its effect for more than 3 h, while the native SBTI showed the effect only within 2 h after i.v . injection.

J Biochem (Tokyo), 1996 Jun, 119(6), 1196 - 201
A new inhibitor of mitochondrial fatty acid oxidation; Hashimoto T et al.; The mitochondrial enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein (trifunctional protein) plays a major role in mitochondrial fatty acid oxidation . The enzyme complex consists of four molecules of alpha-subunit containing both hydratase and dehydrogenase domains and four molecules of beta-subunit containing the thiolase domain . The primary structure of a gastrin-binding protein (GBP) was highly homologous to that of the alpha-subunit of the trifunctional protein . Here, we report that gastrin inhibits the hydratase, dehydrogenase, and thiolase activities of the trifunctional protein . The gastrin/cholecystokinin receptor antagonist benzotript, which inhibited binding of gastrin to the GBP, also inhibited all three activities of the trifunctional protein . In addition, benzotript inhibits the activities of multifunctional enzymes having similar structures, such as the peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein and the Pseudomonas fragi fatty acid oxidation enzyme complex . This reagent, however, hardly inhibited various monofunctional enzymes involved in fatty acid oxidation.

Vaccine, 1996 Jun, 14(8), 817 - 27
Plasmodium falciparum circumsporozoite vaccine immunogenicity and efficacy trial with natural challenge quantitation in an area of endemic human malaria of Kenya; Sherwood JA et al.; It has been hypothesized that antibody induced by Plasmodium falciparum circumsporozoite protein vaccine would be effective against endemic human malaria . In a malaria endemic region of Kenya, 76 volunteers, in 38 pairs sleeping adjacently, were immunized with subunit circumsporozoite protein Asn-Ala-Asn-Pro tetrapeptide repeat-pseudomonas toxin A, or hepatitis B vaccine . After quinine and doxcycycline, volunteers were followed for illness daily, parasitemia weekly, antibody, T-lymphocyte responses, and treated if indicated . Anopheles mosquitoes resting in houses were collected, and tested for P . falciparum antigen, or dissected for sporozoites and tested for blood meal ABO type and P . falciparum antigen . Vaccine was safe, with side-effects similar in both groups, and immunogenic, engendering IgG antibody as high as 600 micrograms ml-1, but did not increase the proportion of volunteers with T-lymphocyte responses . Estimation of P . falciparum challenge averaged 0.194 potentially infective Anopheles bites/volunteer/ day . Mosquito blood meals showed no difference in biting intensity between vaccine and control groups . Both groups had similar malaria-free survival curves, cumulative positive blood slides, cumulative parasites mm-3, and numbers of parasites mm-3 on first positive blood slide, during three post-vaccination observation periods . Every volunteer had P . falciparum parastemia at least once . Vaccinees had 82% and controls 89% incidences of symptomatic parasitemia (P = 0.514, efficacy 9%, statistical power 95% probability of efficacy < 50%) . Vaccine-induced anti-sporozoite antibody was not protective in this study . Within designed statistical precisions the present study is in agreement with efficacy studies in Colombia, Venezuela and Tanzania.

Appl Environ Microbiol, 1996 Jun, 62(6), 2169 - 73
A marine oligobacterium harboring genes known to be part of aromatic hydrocarbon degradation pathways of soil pseudomonads; Wang Y et al.; The far-ranging distribution of genes for aromatic hydrocarbon catabolism, predominantly studied in soil pseudomonads, is extended to a marine oligobacterium by finding five homologous sequences in a 5.7-kb chromosomal DNA from a new isolate, Cycloclasticus oligotrophus RB1 . RB1 is capable of growth in unamended seawater or mineral salts media supplemented with a variety of aromatic compounds, including toluene, o-, m-, or p-xylenes, as sole carbon sources . The five open reading frames, designated xylM, K, G, C1, and C2, are 57% A+T-rich . XylM is predicted to be an integral membrane protein; XylK and XylG possess glutathione S-transferase (GST) and 2-hydroxy-5methyl-6-oxohexa2,4-dienoate dehydrogenase activities, respectively; XylC1C2 are homologs of the large and small subunits of the iron sulfur protein component of the biphenyl dioxygenase (e.g., BphA1A2).

Genetics, 1996 Jun, 143(2), 973 - 82
Isolation of Arabidopsis mutants with enhanced disease susceptibility by direct screening; Glazebrook J et al.; To discover which components of plant defense responses make significant contributions to limiting pathogen attack, we screened a mutagenized population of Arabidopsis thaliana for individuals that exhibit increased susceptibility to the moderately virulent bacterial pathogen Pseudomonas syringae pv . maculicola ES4326 (Psm ES4326) . The 12 enhanced disease susceptibility (eds) mutants isolated included alleles of two genes involved in phytoalexin biosynthesis (pad2, which had been identified previously, and pad4, which had not been identified previously), two alleles of the previously identified npr1 gene, which affects expression of other defense genes, and alleles of seven previously unidentified genes of unknown function . The npr1 mutations caused greatly reduced expression of the PR1 gene in response to PsmES4326 infection, but had little effect on expression of two other defense genes, BGL2 and PR5, suggesting that PR1 expression may be important for limiting growth of PsmES4326 . While direct screens for mutants with quantitative pathogen-susceptibility phenotypes have not been reported previously, our finding that mutants isolated in this way include those affected in known defense responses supports the notion that this type of screening strategy allows genetic dissection of the roles of various plant defense responses in disease resistance.

J Appl Bacteriol, 1996 Jun, 80(6), 589 - 95
Interaction between fish spoilage bacteria Pseudomonas sp . and Shewanella putrefaciens in fish extracts and on fish tissue; Gram L et al.; The interaction between fish spoilage bacteria, Pseudomonas sp . and Shewanella putrefaciens, was investigated using fish extract and fish tissue as model systems . Isolates of Pseudomonas that produced iron chelators, siderophores, inhibited growth of S . putrefaciens in a fish-extract-agar diffusion assay but no, or only weak, antagonistic activity was seen when the medium was supplemented with iron . Sterile-filtered supernatant fluid from a siderophore-producing Pseudomonas grown in fish extract was inhibitory to S . putrefaciens if the number of Psudomonas was above 10(8) cfu ml-1 . In contrast, supernatant fluids from siderophore-negative Pseudomonas isolates did not inhibit growth of S . putrefaciens . The inhibitory effect was, except for one strain of Pseudomonas, not seen in supernatant fluids from iron-enriched cultures of Pseudomonas sp . Finally, siderophore-producing Pseudomonas sp . lowered the maximum cell level of S . putrefaciens 1-2 log units from 10(9) to 10(10) cfu g-1 when the strains were grown on fish muscle blocks at 0 degrees C but the growth rate of S . putrefaciens was not affected.

Arch Microbiol, 1996 Jun, 165(6), 415 - 7
Identification of activities that catalyze the cis-trans isomerization of the double bond of a mono-unsaturated fatty acid in Pseudomonas sp . strain E-3; Okuyama H et al.; A cell-free extract of Pseudomonas sp . strain E-3 catalyzed the conversion of 9-cis-hexadecenoic acid {16:1(9c)} to 9-trans-hexadecenoic acid {16:1(9t)} in the free acid form and when 16:1(9c) was esterified to phosphatidylethanolamine (PE) . The cytosolic fraction catalyzed the isomerizations of free 16:1(9c) by itself and of 16:1(9c) esterified to PE in the presence of the membrane fraction . Tracer experiments using {2,2-2H2}16:1(9c) demonstrated that the isomerization of free 16:1(9c) occurred independently of the isomerization of 16:1(9c) esterified to PE, indicating that this bacterium has two types of activities that catalyze the cis-trans isomerization of the double bond of a mono-unsaturated fatty acid.

J Bacteriol, 1996 Jun, 178(11), 3353 - 6
Stereospecific dihydroxylation of the styrene vinyl group by purified naphthalene dioxygenase from Pseudomonas sp . strain NCIB 9816-4; Lee K et al.; Naphthalene dioxygenase (NDO) from Pseudomonas sp . strain NCIB 9816-4 adds both atoms of the dioxygen molecule to styrene to form (R)-l-phenyl-1,2-ethanediol . Product formation is tightly coupled to dioxygen consumption and NADH oxidation . NDO oxidizes styrene-d8 at almost the same initial rate as styrene . The results indicate that dioxygen activation by NDO is different from that by cytochrome P-450 and other monooxygenases, which oxidize styrene to styrene 1,2-oxide.

Ann Thorac Surg, 1996 Jun, 61(6), 1797 - 804
Clinical improvement after revision in Fontan patients; Vitullo DA et al.; BACKGROUND . Arrhythmias, decreased exercise tolerance, or malabsorption will develop in a significant number of Fontan patients . Fontan revision consisting of creation of lateral atrial tunnel, reconnection of the Glenn shunt when present, or both appears to improve these patients . METHODS . Over a 34-month period, 9 patients underwent Fontan revision . The mean age was 11 +/- 5 years and the mean interval from Fontan operation to revision was 3 +/- 2 years . The reason for revision included marked impairment in exercise capacity, inability to go to school consistently, and chronic fatigue in 6 patients, 3 of whom also had serious atrial arrhythmias . Five of the 6 patients had a classic Glenn shunt . The mean right atrial pressure was greater than the pressure of the Glenn shunt (20 +/- 1.6 versus 17 +/- 0.8 mm Hg) . Three of the 6 patients also showed a significant gradient between the right or left pulmonary artery wedge and ventricular end-diastolic pressure, indicating pulmonary vein obstruction from the bulging atrial septum or partitioning patch (13 +/- 3 versus 6.8 +/- 1 mm Hg) . The remaining 3 patients had revision because of malabsorption (1), hepatomegaly and obstructed right pulmonary veins from bulging atrial septum (1), and tricuspid insufficiency (1) . Fontan revision was accomplished with creation of a lateral atrial tunnel and Glenn reconnection in 6 patients, Glenn reconnection in 2, and creation of a lateral atrial tunnel in 1 . Four patients had additional procedures . RESULTS . One patient died of Pseudomonas pneumonia . Early extubation, chest tube removal, and postoperative hospital discharge were accomplished in 8 patients (mean = 1.4 +/- 1, 2.8 +/- 1, and 8 +/- 3 days, respectively) . One patient died 8 months postoperatively of brain damage after ventricular fibrillation from attempted cardioversion for atrial flutter . The remaining patients had marked improvement in exercise capacity with ability to consistently go to school, improvement in duration and tolerance to arrhythmias on less medication, and resolution of malabsorption up to 37 months postoperatively (mean, 20 +/- 12 months) . CONCLUSIONS . We conclude that creation of lateral atrial tunnel with excision of a bulging atrial septum or atrial partitioning patch that causes pulmonary venous obstruction, reconnection of the Glenn shunt, which allows better distribution of flow based on the pulmonary vascular bed and resistance of each lung, or a combination of these procedures will improve Fontan patients.

Biochemistry, 1996 May 28, 35(21), 6891 - 9
Role of CAS, a human homologue to the yeast chromosome segregation gene CSE1, in toxin and tumor necrosis factor mediated apoptosis; Brinkmann U et al.; We have previously isolated by expression/selection cloning plasmids containing human cDNAs that rendered MCF-7 breast cancer cells resistant to immunotoxins, Pseudomonas exotoxin (PE), and diphtheria toxin (DT) {Brinkmann et al . (1995) Mol . Med . 1, 206-216} . Here we describe that one of these resistant plasmids, which contains an antisense cDNA fragment homologous to the yeast chromosome segregation gene CSE1 {CAS; Brinkmann et al . (1995) Proc . Natl . Acad . Sci . U.S.A . 92, 10427-10431}, reduces the intracellular content of the human CSE1 homologue CAS protein . CAS reduction confers resistance not only to the ADP-ribosylating toxins PE and DT, but also to tumor necrosis factor alpha and beta . The resistance was observed as reduced apoptosis . CAS antisense did not affect the cell death induced by staurosporine, cycloheximide, or etoposide . The observation that CAS antisense can interfere with apoptosis mediated by TNF and ADP-ribosylating toxins suggests that CAS may play a role in selected pathways of apoptosis.

Blood, 1996 May 15, 87(10), 4333 - 9
Targeting of interleukin-13 receptor on human renal cell carcinoma cells by a recombinant chimeric protein composed of interleukin-13 and a truncated form of Pseudomonas exotoxin A (PE38QQR); Puri RK et al.; We have previously shown that human renal cell carcinoma (RCC) cells express large numbers of interleukin-13 receptors (IL-13R), a newly described hemopoietic growth factor receptor . To target tumor cells that express IL-13R, we have produced a chimeric protein composed of human IL-13 and a derivative of Pseudomonas exotoxin A, termed PE38QQR . We report here that IL13-PE38QQR is highly cytotoxic to many human RCC cell lines . IL-13R-negative cell lines or cell lines expressing low numbers of IL-13R ( < 300 sites/cell) that include human bone marrow-derived cells were not susceptible to the cytotoxic effect of IL 13-PE38QQR . The sensitivity of RCC cells to IL13-PE38QQR correlated positively with the density of IL-13R . The cytotoxic activity of IL13-PE38QQR was competed by an excess of IL-13 in a protein synthesis inhibition assay and confirmed by a clonogenic assay . Even though IL-13 and IL-4 are homologues and IL-4R and IL-13R have been proposed to share a receptor subunit, IL-4 did not compete for the cytotoxicity mediated by IL13-toxin on RCC . IL13-PE38QQR competes for {125I}-IL-13 binding sites on RCC cells, although at a lower affinity than the wild-type recombinant cytokine . Human T-cell, B-cell, and monocytic cell lines are unresponsive to the cytotoxic action of IL13-PE38QQR . Thus, our results indicate that IL13-PE38QQR is highly cytotoxic to human RCC cells, although it is not cytotoxic to a variety of normal hematopoietic cells . IL13-PE38QQR should be further investigated preclinically for the treatment of human RCCs.

Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 5099 - 104
Ozone-induced responses in Arabidopsis thaliana: the role of salicylic acid in the accumulation of defense-related transcripts and induced resistance; Sharma YK et al.; Exposure of Arabidopsis thaliana to ozone results in the expression of a number of defense-related genes that are also induced during a hypersensitive response . A potential common link between the activation of defense gene expression during a hypersensitive response and by ozone treatment is the production of active oxygen species and the accumulation of hydrogen peroxide . Here we report that salicylic acid accumulation, which can be induced by hydrogen peroxide and is required for the expression of both a hypersensitive response and systemic acquired resistance, is also required for the induction of some, but not all, ozone-induced mRNAs examined . In addition, we show that ozone exposure triggers induced resistance of A . thaliana to infection with virulent phytopathogenic Pseudomonas syringae strains . Infection of transgenic plants expressing salicylate hydroxylase, which prevents the accumulation of salicylic acid, or npr1 mutant plants, which are defective in the expression of systemic acquired resistance at a step downstream of salicylic acid, demonstrated that the signaling pathway activated during ozone-induced resistance overlaps with the systemic acquired resistance activation pathway and is salicylic acid dependent . Interestingly, plants expressing salicylate hydroxylase exhibited increased sensitivity to ozone exposure . These results demonstrate that ozone activates at least two distinct signaling pathways, including a salicylic acid dependent pathway previously shown to be associated with the activation of pathogen defense reactions, and that this latter pathway also induces a protective response to ozone.

Biochemistry, 1996 May 14, 35(19), 6020 - 5
Three-dimensional structure of the zinc-containing phosphotriesterase with the bound substrate analog diethyl 4-methylbenzylphosphonate; Vanhooke JL et al.; Phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of paraoxon and related acetylcholinesterase inhibitors with rate enhancements that approach 10(12) . The enzyme requires a binuclear metal center for activity and as isolated contains 2 equiv of zinc per subunit . Here we describe the three-dimensional structure of the Zn2+/Zn2+-substituted enzyme complexed with the substrate analog diethyl 4-methylbenzylphosphonate . Crystals employed in the investigation belonged to the space group C2 with unit cell dimensions of a = 129.6 A, b = 91.4 A, c = 69.4 A, beta = 91.9 degrees, and two subunits in the asymmetric unit . The model was refined by least-squares analysis to a nominal resolution of 2.1 A and a crystallographic R-factor of 15.4% for all measured X-ray data . As in the previously reported structure of the cadmium-containing enzyme, the bridging ligands are a carbamylated lysine residue (Lys 169) and a hydroxide . The zinc ions are separated by 3.3 A . The more buried zinc ion is surrounded by His 55, His 57, Lys 169, Asp 301, and the bridging hydroxide in a trigonal bipyramidal arrangement as described for the cadmium-substituted enzyme . Unlike the octahedral coordination observed for the more solvent-exposed cadmium ion, however, the second zinc is tetrahedrally ligated to Lys 169, His 201, His 230, and the bridging hydroxide . The diethyl 4-methylbenzylphosphonate occupies a site near the binuclear metal center with the phosphoryl oxygen of the substrate analog situated at 3.5 A from the more solvent-exposed zinc ion . The aromatic portion of the inhibitor binds in a fairly hydrophobic pocket . A striking feature of the active site pocket is the lack of direct electrostatic interactions between the inhibitor and the protein . This most likely explains the broad substrate specificity exhibited by phosphotriesterase . The position of the inhibitor within the active site suggests that the nucleophile for the hydrolysis reaction is the metal-bound hydroxide.

Gene, 1996 May 8, 170(2), 213 - 6
Nucleotide sequences of four pathogen-induced alfalfa peroxidase-encoding cDNAs; el-Turk J et al.; We constructed an alfalfa cDNA library from mRNA extracted from leaves after infection with Pseudomonas syringae (incompatible interaction) . Screening with oligodeoxyribonucleotides designed from regions conserved in all known peroxidases allowed the isolation of four cDNAs (Msprx1A, 1B, 1C and 2) . Sequence analysis revealed the presence of open reading frames of 351, 355, 358 and 323 amino acids, respectively, with the characteristic consensus sequences of plant peroxidases . Sequence comparison showed that the Msprx2 product is significantly different from the others and, particularly, lacks a C-terminal propeptide which might be required for vacuolar targeting.

J Biol Chem, 1996 May 3, 271(18), 10560 - 8
Target cell-specific DNA transfer mediated by a chimeric multidomain protein . Novel non-viral gene delivery system; Fominaya J et al.; Based on the multidomain structure of the bacterial Pseudomonas exotoxin A, a recombinant fusion protein was constructed which serves as a target cell-specific carrier for the transfer of DNA via receptor-mediated endocytosis . The protein consists of three functional domains: 1) an ErbB-2 -specific single chain antibody confers target cell specificity, 2) the exotoxin A translocation domain facilitates endosome escape, and 3) a DNA binding domain derived from the yeast GAL4 protein enable sequence-specific high affinity binding to DNA . Carrier protein purified from bacterial lysates displayed both ErbB-2-specific and DNA sequence-specific binding in vitro . Complexes which formed spontaneously by the interaction of the fusion protein with a luciferase reporter gene construct carrying a GAL4-specific recognition sequence, after condensation of the DNA and compensation of excess negative charge with poly-L-lysine were able to transfect ErbB-2-expressing cells in vitro in a cell-specific manner . Transient expression of the luciferase gene driven by the SV40 early promoter was observed and correlates with the amount of carrier protein in the complex . Truncated forms of the carrier protein lacking either the cell recognition domain or the translocation domain failed to facilitate efficient DNA transfer.

Bone Marrow Transplant, 1996 May, 17(5), 793 - 9
Specific depletion of alloreactivity against haplotype mismatched related individuals by a recombinant immunotoxin: a new approach to graft-versus-host disease prophylaxis in haploidentical bone marrow transplantation; Mavroudis DA et al.; Haploidentical bone marrow transplantation (BMT) is associated with a high risk of severe graft-versus-host disease (GVHD) . While pan-T cell depletion of the graft is the most effective means of preventing severe GVHD, it is associated with delayed recovery of T cell function leading to fatal infections . We used two related Pseudomonas exotoxin-based immunotoxins, anti-Tac(Fv)-PE38 and anti-Tac(Fv)-PE38KDEL, that both target the IL-2 receptor on activated T cells, to specifically deplete alloreactive lymphocytes against haploidentical stimulators . The functional capacity of the remaining lymphocytes was tested in proliferative assays against the original haploidentical stimulator and pooled cells from other mismatched donors (third party) . We varied the recombinant toxin concentration and schedule to determine the optimum conditions for selective depletion . In 10 experiments, the mean residual reactivity after depletion was 7.6 +/- 1.4% against the haploidentical stimulator and 64.2 +/- 5% against the third party, expressed as a percentage of the undepleted response to the same stimulators . Depletion was shown to be specific for mixed lymphocyte culture (MLC)-activated lymphocytes . The immunotoxin did not affect CFU-GM growth of normal BM cells . This selective depletion of haploidentical alloreactivity could be used to prevent GVHD while conserving immune recovery following haploidentical BMT.

Microbiology, 1996 May, 142 ( Pt 5), 1191 - 9
Acquisition of iron by the non-siderophore-producing Pseudomonas fragi; Champomier-Verges MC et al.; The iron requirement, siderophore production and iron uptake mechanisms of the type strain Pseudomonas fragi ATCC 4973 and five P . fragi isolates from meat were analysed . The strains exhibited a high sensitivity to iron starvation: their growth was strongly inhibited in medium supplemented with the iron chelator ethylenediamine di(hydroxyphenylacetic acid) or in medium treated with 8-hydroxyquinoline to remove contaminating iron . No siderophores were detectable in the growth supernatants of iron-starved cells . Cross-feeding experiments in iron-depleted medium showed, however, that the bacterial growth could be strongly stimulated by siderophores of foreign origin including desferriferrioxamine B, enterobactin and some pyoverdines . Moreover, all the strains were capable of efficiently using the iron sources present in their natural environment, i.e., transferrin, lactoferrin and haemoglobin . Iron starvation led to the specific production of supplementary outer-membrane proteins of apparent molecular mass ranging from 80 to 88 kDa . Furthermore, growth in the presence of exogenous siderophores resulted, in some strains, in the induction of siderophore-mediated iron uptake systems . For one strain the concomitant synthesis of an iron-regulated, siderophore-inducible outer-membrane protein was observed.

PDA J Pharm Sci Technol, 1996 May-Jun, 50(3), 147 - 53
Evaluation of recovery filters for use in bacterial retention testing of sterilizing-grade filters; Carter J; Membrane filters with pore-size ratings of 0.22 microns and 0.45 microns were tested for their ability to recover Pseudomonas diminuta ATCC 19146 (P . diminuta), the organism typically used in bacterial retention testing of sterilizing-grade membrane filters . For each of the two pore-size ratings, filters of two membrane filter polymer materials, hydrophilic PVDF (Millipore Durapore) and mixed esters of cellulose, were tested, resulting in an evaluation of four potential recovery filters . The 0.45 microns mixed esters of cellulose filter is the currently accepted membrane for this purpose . The data show no difference in the ability of the four filters to recover freshly cultured P . diminuta . Moreover, the membrane-filter method was shown to provide a very high bacterial-recovery efficiency, equivalent to that of the spread-plate method . Thus, 0.22 micron filters, despite their ability to retain higher levels of bacteria, proved not to have an advantage over 0.45 micron membranes in terms of bacterial recovery . This result, combined with (1) the knowledge that more open membranes have been shown experimentally to more efficiently recover stressed organisms; (2) the potential to produce stressed cells in an actual bacterial retention test; and (3) the long history of the successful use of 0.45 microns mixed esters of cellulose for bacterial recovery, support the continued use of the 0.45 micron filter in this application.

Mol Plant Microbe Interact, 1996 May, 9(4), 272 - 81
Cloning and characterization of tek, the gene encoding the major extracellular protein of Pseudomonas solanacearum; Denny TP et al.; Susceptible plants infected by Pseudomonas solanacearum usually will, largely due to extracellular proteins (EXPs) and the high-molecular-mass extracellular polysaccharide (EPS I) this pathogen produces . Circumstantial evidence suggested that a 28-kDa protein, the single most abundant EXP made by P . solanacearum in culture, is associated with production of EPS I, and thus might have a role in pathogenesis . The 28-kDa EXP was purified and, based on its N-terminal amino acid sequence, an oligonucleotide mixture was made and used as a hybridization probe to clone the gene encoding it . DNA sequence analysis suggested that the coding sequence for the 28-kDa EXP is within a gene, designated tek, that encodes a 58-kDa membrane-associated precursor protein that is processed by signal peptidase II during export . Analysis of radiolabeled polypeptides expressed from tek confirmed that it encodes a 58-kDa precursor protein, which is exported out of the cells as a 55-kDa preprotein and processed extracellularly to release the very basic 28-kDa EXP from its C terminus . The position, transcriptional direction, and regulated expression of tek suggest that it is cotranscribed with xpsR, a gene essential for regulating biosynthesis of EPS I, and reinforces the association of the 28-kDa EXP with virulence . However, since P . solanacearum mutants lacking only the 28-kDa EXP produced wild-type amounts of EPS I and were fully virulent, the function of this protein remains unclear.

J Invest Dermatol, 1996 May, 106(5), 1075 - 80
Lysozyme binds to elastin and protects elastin from elastase-mediated degradation; Park PW et al.; Lysozyme has been shown to be associated with damaged elastic fibers in many tissues and organs . To better characterize this interaction, binding of lysozyme to elastin was studied using solution-based binding assays . Under physiologic conditions, radio-labeled lysozyme bound specifically to elastin in a time- and concentration-dependent manner . Binding was reversible and was inhibited by unlabeled human and hen lysozyme but not by other proteins . Lysozyme had no elastolytic activity as assessed by a standard tritium-release assay, but, importantly, prevented the proteolytic degradation of elastin by human leukocyte elastase, pancreatic elastase, thermolysin, and Pseudomonas elastase . A striking feature of lysozyme's anti-elastase activity was that it did not function in the classical sense of inhibiting directly the enzymatic activity of the protease . Instead, by binding to elastin, lysozyme prevented the protease from interacting with the elastin substrate in ways that normally favor proteolysis . These results show that lysozyme binds to the elastin component of elastic fibers and that this interaction has important biological consequences for elastic fiber degradation . By preventing degradation of elastin, lysozyme can function as an important natural inhibitor that exerts a protective effect on elastic fibers at sites of tissue injury.

Biochem Biophys Res Commun, 1996 Apr 25, 221(3), 631 - 5
Enhancement of thermostability and catalytic efficiency of AprP, an alkaline protease from Pseudomonas sp., by the introduction of a disulfide bond; Ko JH et al.; A site-directed mutagenesis in AprP, an alkaline protease isolated from Pseudomonas sp . KFCC 10818 was carried out in order to obtain increased thermostability . Sites for cysteine substitutions to form disulfide bond within AprP were chosen by comparing the sequences with aqualysin I, an alkaline thermostable serine protease whose disulfide bonds seems to be important for its thermostability . Gly199 and Phe236 residues were each replaced with cysteine by site-directed mutagenesis . The G199C/F236C mutant enzyme appeared to form a disulfide bond spontaneously during its expression . It also showed improved kinetic parameters for the hydrolysis of a synthetic peptide substrate at pH 8.5 and 10.5 compared to those of the wild-type enzyme . The half-life of the G199C/F236C mutant was found to be 2 to 4.8 times longer than that of wild-type under various experimental conditions, except when tested under reducing condition, where no significant differences in the half-life of the two types were observed . Therefore, it is concluded that the introduction of the disulfide bond enhanced the thermostability and the catalytic efficiency of the enzyme AprP.

Eur J Biochem, 1996 Apr 15, 237(2), 447 - 53
Mutation of the conserved Cys165 outside of the CuA domain destabilizes nitrous oxide reductase but maintains its catalytic activity . Evidence for disulfide bridges and a putative protein disulfide isomerase gene; Dreusch A et al.; The single conserved Cys165 outside of the CuA domain of nitrous oxide reductase (N2OR) from Pseudomonas stutzeri was mutated to glycine to test its presumed function in metal coordination of the catalytic site, CuZ . The point mutation reduced the cellular level of N2OR 5--10-fold compared to the level of the control strain . In the mutant, the activity and the Cu content of the enzyme, as well as the transcript level of the N2OR structural gene, nosZ, remained unaffected . The mutant enzyme was processed and exported into the periplasm like the wild-type enzyme . Chemical analysis for sulfhydryl groups gave about nine -SH groups/monomer of the apoenzyme prepared from the wild-type enzyme, in accordance with the nine cysteine residues of the derived amino acid sequence . Eight -SH groups were found to form disulfide bridges in the holoenzyme dimer . We propose that in the native state of the enzyme Cys165 does not bind to CuZ, but may be part of a disulfide bridge essential for the stability of N2OR . Immediately downstream of the genes nosDFY, encoding the components for Cu incorporation into the reductase, we have identified the open reading frame, ORFL, whose derived product has the signature of a protein disulfide isomerase.

Cell Immunol, 1996 Apr 10, 169(1), 55 - 61
Interleukin 2 pseudomonas exotoxin (IL2-PE66(4)Glu) chimeric protein kills B cells from patients with Myasthenia gravis; Steinberger I et al.; IL2-PE66(4)Glu is a chimeric cytotoxin consisting of interleukin 2 (IL2) fused to a mutant form of Pseudomonas exotoxin (PE66(4)Glu) . The chimeric cytotoxin has been previously shown to be extremely toxic to both phytohemagglutinin blasts and mixed leukocyte reaction blasts prepared from monkey and human lymphocytes . To explore the possible clinical utility of IL2-PE66(4)Glu for autoimmune diseases, particularly in which B cells are involved, we tested fresh B cells from patients with myasthenia gravis for sensitivity to this chimeric cytotoxin . Seventy-six percent (16 of 21) of the B cells tested were markedly sensitive to IL2-PE66(4)Glu-mediated cytotoxicity, with inhibition of protein synthesis ranging from 20 to 92% . B cells from control donors were much less sensitive to IL2-PE66(4)Glu cytotoxicity . Moreover, a control protein lacking IL2 as the targeting moiety of the chimera had no effect toward all B cells tested, thus establishing its specific activity . Our results suggest that IL2-PE66(4)Glu could be an effective tool for selective targeted immunotherapy of myasthenia gravis patients.

Lett Appl Microbiol, 1996 Apr, 22(4), 275 - 9
2-Chlorobenzoic acid and 2,5-dichlorobenzoic acid metabolism by crude extracts of Pseudomonas sp . CPE2 strain; Fava F et al.; Crude extracts of Pseudomonas sp . CPE2 strain, which is capable of growing on 2-chlorobenzoic acid (2-CBA) and 2,5-dichlorobenzoic acid (2,5-dCBA) in the absence of other carbon sources, were found to be capable of bioconverting 2-CBA and 2,5-dCBA to catechol and 4-chlorocatechol, respectively, by a reaction requiring molecular oxygen and exogenous NADH . Extracts obtained from 2-CBA-grown cells in the presence of 2-CBA and from 2,5-dCBA-grown cells in the presence of 2,5-dCBA were found to have activities similarly influenced by the assay parameters pH, temperature, and by concentration of oxygen, protein, Fe2+, FAD and NADH in the assay medium . In addition, the activity of the two crude extracts in the presence of 2-CBA or 2,5-dCBA was described by very similar Michaelis-Menten kinetic parameters . These observations led to the speculation that a unique broad-spectrum chlorobenzoate 1,2-dioxygenase catalyses the 2-CBA and 2,5-dCBA metabolism both in 2-CBA- and 2,5-dCBA-grown CPE2 cells.

Enzyme Microb Technol, 1996 Apr, 18(5), 353 - 7
Chemoenzymatic synthesis of D(-)phenylglycine using hydantoinase of Pseudomonas desmolyticum resting cells; Gokhale DV et al.; We screened 125 Pseudomonas strains from our culture collection for the production of hydantoinase activity using DL-phenylhydantoin as a substrate . Pseudomonas desmolyticum NCIM 2112 was found to be the best hydantoinase (dihydropyrimidinase E.C . 3.5.2.2) producer . The enzymatic reactions were carried out using 18-20-h grown cells in nutrient broth and 5-phenylhydantoin as the substrate . Optimization studies for the biotransformation reaction were performed to increase product yield . The optimum pH and temperature for D(-)N-carbamoylphenylglycine production were 9.5 and 30 degrees C, respectively . Biotransformation under these alkaline conditions allowed the complete conversion of 27.0 g l-1 of DL-phenylhydantoin to 26.5 g l-1 of N-carbamoylphenylglycine within 24 h, with a molar yield of 90% . The hydantoinase involved in this biotransformation process was strictly D-stereospecific, because the product isolated was pure D(-)N-carbamoylphenylglycine . This pure product was further chemically converted to D(-)phenylglycine using nitrous acid with an 80% chemical yield . Thus, the overall conversion efficiency of DL-5-phenylhydantoin to D(-)phenylglycine was found to be 65-68%.

Proteins, 1996 Apr, 24(4), 520 - 2
Crystallization and preliminary x-ray crystallographic studies of L-2-haloacid dehalogenase from Pseudomonas sp . YL; Hisano T et al.; The dimeric L-2-haloacid dehalogenase from Pseudomonas sp . YL, (subunit mass, 26179 Da), has been crystallized by vapor diffusion, supplemented by repetitive seeding, against a 50 mM potassium dihydrogenphosphate solution (pH 4.5) containing 15% (w/v) polyethylene glycol 8,000 and 1% (v/v) n-propanol . The crystals belong to the monoclinic space group C2 with unit cell dimensions of a = 92.21 angstrom, b = 62.78 angstrom, c = 50.84 angstrom, and beta = 122.4 degrees, and contain two dehalogenase dimers in the unit cell . They are of good quality and diffract up to 1.5 angstrom resolution.

Biosci Biotechnol Biochem, 1996 Apr, 60(4), 650 - 3
Chemical modification of histidine residue of N-acyl-D-Glutamate amidohydrolase from Pseudomonas sp . 5f-1; Wakayama M et al.; N-Acyl-D-glutamate amidohydrolase (D-AGase) from Pseudomonas sp . 5f-1 was inactivated by diethyl pyrocarbonate (DEP) . The chemical modification by DEP showed a difference spectrum at 246 nm due to the N-carbethoxyhistidine residue . Removal of the carbethoxy group from inactivated enzyme with hydroxylamine restored enzyme activity . The inactivation by DEp proceeded with pseudo-first-order kinetics, and was protected in the presence of the substrate N-acetyl-D-glutamate (Glu), or the competitive inhibitor sodium alpha-ketoglutarate (alpha-KGA) . These results suggest the presence of an essential histidine residue at or near of the active site of the enzyme.

Biosci Biotechnol Biochem, 1996 Apr, 60(4), 612 - 5
N-carbamyl-L-amino acid amidohydrolase of Pseudomonas sp . strain NS671: purification and some properties of the enzyme expressed in Escherichia coli; Ishikawa T et al.; An N-carbamyl-L-amino acid amidohydrolase was purified from cells of Escherichia coli in which the gene for N-carbamyl-L-amino acid amidohydrolase of Pseudomonas sp . strain NS671 was expressed . The purified enzyme was homogeneous by the criterion of SDS-polyacrylamide gel electrophoresis . The results of gel filtration chromatography and SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a dimeric protein with 45-kDa identical subunits . The enzyme required Mn2+ ion (above 1 mM) for the activity . The optimal pH and temperature were 7.5 and around 40 degrees C, respectively, with N-carbamyl-L-methionine as the substrate . The enzyme activity was inhibited by ATP and was lost completely with p-chloromercuribenzoate (1 mM) . The enzyme was strictly L-specific and showed a broad substrate specificity for N-carbamyl-l-alpha-amino acids.

Eur J Epidemiol, 1996 Apr, 12(2), 149 - 53
Field inversion gel electrophoresis on Pseudomonas cepacia strains isolated from cystic fibrosis patients; Amalfitano G et al.; Genome fingerprinting by field inversion gel electrophoresis (FIGE) was utilized to typify 129 isolates of Pseudomonas cepacia (Pc) from 59 patients with cystic fibrosis (CF) and from environmental cultures in the CF ward . The aim of this study was to assess whether a segregation policy avoided colonization of CF patients by nosocomial strains and contamination of the environment by colonized individuals, whether or not an 'epidemic strain' was present in the ward and whether cross-colonization occurred in CF individuals subjected to prolonged close contact . The Pc strains of each patient remained unchanged over time; 78% of the genome finger printings (GFP) were individual, whereas the others gave rise to 9 GFP groups . A spirometer was probably contaminated by a newly colonized patient . Adequate sanitary measures and avoidance of excessive promiscuity are helpful for limiting but are unable to eliminate Pc transmission in the CF ward . Direct or indirect transmission, however seems, more frequent in CF patients in contacts outside the hospital.

J Pediatr Surg, 1996 Apr, 31(4), 594 - 5
Aortoesophageal fistula and double aortic arch: two important points in management; Othersen HB Jr et al.; Two children with double aortic arch and aortoesophageal fistula (AEF) are reported to warn of this lethal complication of double aortic arch and to stress important points in the diagnosis and management . A review of the records of 30 children with double aortic arch disclosed two patients who had AEF . The first patient had respiratory distress and repair of a vascular ring (double aortic arch) at 5 weeks of age . At 9 weeks of age, because of difficulty with tracheal extubation, aortopexy was performed . Ten days later, profuse upper gastrointestinal bleeding required control by a Sengstaken-Blakemore (SB) tube . Thoracotomy and repair AEF was accomplished successfully under cardiopulmonary bypass . The second patient had hepatomegaly and Pseudomonas sepsis . Endotracheal and nasogastric intubation was necessary, and subsequently the double aortic arch was demonstrated by magnetic resonance imaging (MRI) . On the 48th day of hospitalization, life-threatening upper gastrointestinal hemorrhage required insertion of an SB tube . Cardiopulmonary bypass allowed successful repair of the AEF . Both children are alive, after 3 and 2 years (respectively) . These patients demonstrate that AEF must be diagnosed clinically (no imaging technique is effective); its history and physical presentation are typical . The SB tube is effective for controlling the hemorrhage until cardiopulmonary bypass can be performed to allow repair.

Curr Biol, 1996 Apr 1, 6(4), 427 - 37
Calcium-mediated apoptosis in a plant hypersensitive disease resistance response; Levine A et al.; BACKGROUND: Avirulent pathogens elicit a battery of plant defenses, often accompanied by collapse of the challenged cells . In soybean cells, sustained accumulation of H2O2 from an oxidative burst cues localized host cell death . Such hypersensitive cell death appears to be an active process, but little is known about the mechanisms underlying cellular collapse . RESULTS: We show that H2O2 stimulates a rapid influx of Ca2+ into soybean cells, which activates a physiological cell death program resulting in the generation of large (approximately 50 kb) DNA fragments and cell corpse morphology--including cell shrinkage, plasma membrane blebbing and nuclear condensation--characteristic of apoptosis . In contrast, H2O2 induction of the cellular protectant gene glutathione S-transferase is Ca(2+)-independent . Apoptosis in soybean cells and leaf tissue was induced by avirulent Pseudomonas syringae pv . glycinea but was not observed at comparable stages of the compatible interaction with the isogenic virulent strain, which fails to elicit a hypersensitive response . Apoptosis was also observed at the onset of the hypersensitive response in Arabidopsis leaves inoculated with avirulent P . syringae pv . tomato and in tobacco cells treated with the fungal peptide cryptogein, which is involved in the induction of non-host resistance to Phytophthora cryptogea . CONCLUSIONS: These observations establish a signal function for Ca2+ downstream of the oxidative burst in the activation of a physiological cell death program in soybean cells that is similar to apoptosis in animals . That the characteristic cell corpse morphology is also induced in Arabidopsis and tobacco by different avirulence signals suggests that apoptosis may prove to be a common, but not necessarily ubiquitous, feature of incompatible plant-pathogen interactions . Emerging similarities between facets of hypersensitive disease resistance and the mammalian native immune system indicate that apoptosis is a widespread defence mechanism in eukaryotes.

Plant Mol Biol, 1996 Apr, 31(1), 175 - 81
Molecular characterization of an adenylate cyclase gene of the cyanobacterium Spirulina platensis; Yashiro K et al.; A cyaA gene, encoding an adenylate cyclase, was isolated from a filamentous cyanobacterium, Spirulina platensis, by functional complementation of a cya mutant of Escherichia coli, defective in adenylate cyclase activity . The predicted gene product of cyaA contains a signal peptide-like domain, a putative sensor domain similar to the gene product of vsrA of Pseudomonas solanacearum, a putative membrane-spanning domain and an adenylate cyclase-like catalytic domain . Two other positive clones that complemented the E . coli mutant were isolated from the same cyanobacterium, suggesting that several cya genes are functioning in S . platensis.

Chemosphere, 1996 Apr, 32(8), 1477 - 83
Aroclor 1221 aerobic dechlorination by a bacterial co-culture: role of chlorobenzoic acid degrading bacteria in the process; Fava F; A bacterial co-culture, ECO3, constituted by a polychlorobiphenyl degrading bacterium, Pseudomonas sp . strain CPE1, and two chlorobenzoic acid degrading bacteria, could grow on Aroclor 1221 (75 mg/L) as the sole carbon source without accumulating chlorinated aromatic metabolites in the medium; 44.5% of the Aroclor 1221 organic chlorine was detected as chloride ion in the medium after 115 h of incubation in batch condition . When glass beads (diameter = 3 mm, 30% w/v) or Triton X-100 (0.066% v/v) were added to ECO3 cultures, average dechlorination percentages were 80% and 89.5%, respectively, after the same incubation time . These percentages were significantly higher than those previously observed with the only polychlorobiphenyl degrading member of ECO3, CPE1 strain, in the same culture conditions . This result can be ascribed to the capability of the ECO3 chlorobenzoic acid degrading bacteria of completely mineralizing the chlorinated benzoic acids produced during the Aroclor 1221 degradation . The depletion of these intermediates seems to prevent toxic or inhibitory effects on the bacteria thus permitting a larger Aroclor 1221 metabolization.

Chemosphere, 1996 Apr, 32(8), 1469 - 75
The presence of glass beads or Triton X-100 in the medium enhances the aerobic dechlorination of Aroclor 1221 in Pseudomonas sp . CPE1 culture; Fava F; The culture pure Pseudomonas sp . CPE1 strain capable of metabolizing low-chlorinated biphenyls in the presence of biphenyl was found to be able to grow on Aroclor 1221 in the absence of an additional carbon source . The presence of glass beads (diameter = 3 mm, 30% w/v) or Triton X-100 (0.066% v/v) in the culture medium significantly enhanced the aerobic dechlorination of the polychlorinated biphenyls present in Aroclor 1221 in batch cultures of CPE1 strain . This result has been ascribed to an increase of Aroclor 1221 bioavailability in the cultures containing glass beads or Triton X-100, probably deriving from a greater interface area PCB-water, i.e . the surface area on which the polychlorobiphenyl degradation seems to take place.

Arch Microbiol, 1996 Apr, 165(4), 258 - 64
Adaptation of Pseudomonas sp . GJ1 to 2-bromoethanol caused by overexpression of an NAD-dependent aldehyde dehydrogenase with low affinity for halogenated aldehydes; Van der Ploeg JR et al.; Pseudomonas sp . GJ1 is able to grow with 2-chloroethanol as the sole carbon and energy source, but not with 2-bromoethanol, which is toxic at low concentrations (1 mM) . A mutant that could grow on 2-bromoethanol with a growth rate of 0.034 h-1 at concentrations up to 5 mM was isolated and designated strain GJ1M9 . Measurement of enzyme activities showed that mutant and wild-type strains contained a PMS-linked alcohol dehydrogenase that was active with halogenated alcohols and that was threefold overexpressed in the mutant when grown on 2-chloroethanol, but only slightly overproduced when grown on 2-bromoethanol . Both strains also contained an NAD-dependent alcohol dehydrogenase that had no activity with halogenated alcohols . Haloacetate dehalogenase levels were similar in the wild-type and the mutant . Activities of NAD-dependent aldehyde dehydrogenase were only slightly higher in extracts of the mutant grown with 2-bromoethanol than in those of the wild-type grown with 2-chloroethanol . SDS-PAGE, however, showed that this enzyme amounted to more than 50% of the total cellular protein in extracts of the mutant from 2-bromoethanol-grown cells, which was fourfold higher than in extracts of the wild-type strain grown on 2-chloroethanol . The enzyme was purified and shown to be a tetrameric protein consisting of subunits of 55 kDa . The enzyme had low Km values for acetaldehyde and other non-halogenated aldehydes (0.8-4 microM), but much higher Km values for chloroacetaldehyde (1.7 mM) and bromoacetaldehyde (10.5 mM), while V(max) values were similar for halogenated and non-halogenated aldehydes . Cultures that were pregrown on 2-chloroethanol rapidly lost aldehyde dehydrogenase activity after addition of 2-bromoethanol and chloroamphenicol, which indicates that bromoacetaldehyde inactivates the enzyme . To achieve growth with 2-bromoethanol, the high expression of the enzyme thus appears to be necessary in order to compensate for the high Km for bromoacetaldehyde and for inactivation of the enzyme of bromoacetaldehyde.

J Surg Res, 1996 Apr, 62(1), 17 - 22
Control of hypertrophic scar growth using antibody-targeted photolysis; Wolfort SF et al.; Hypertrophic scar is marked by excess collagen accumulation secondary to an increased vascularization response in the scar and an increase in fibroblast cell density . It is currently the most debilitating long-term complication of the surviving burn patient, and at present, there is no routinely effective form of therapy . In this study, we investigated the potential use of antibody-targeted photolysis (ATPL) in treating hypertrophic scars . An immunoconjugate consisting of a photosensitizer (Sn-chlorin e6) linked to a monoclonal antibody that binds to human myofibroblasts (PR2D3) was prepared, which in response to photoactivation produces singlet oxygen in close proximity to the target cell surface . The model used for these studies consisted of 1-mm 3 human hypertrophic scar tissue implants in athymic mice . These implants increase approximately 20-fold in volume over a period of 15 days . Four days after implantation immunoconjugate was injected directly into scar implants allowed to diffuse throughout for 24 hr before implants were illuminated with laser light at 630 nm (120 J/cm 2) . ATPL treatment caused a significant reduction in total growth compared to the untreated controls (P < 0.05) . No effect was observed when an irrelevant conjugate (anti-Pseudomonas aeruginosa) was used . Histological examination of the ATPL-treated implants 24 hr post-ATPL revealed the presence of a large number of lipid droplets indicative of massive cell damage and infiltration by mononuclear cells and neutrophils.

J Biol Chem, 1996 Mar 29, 271(13), 7705 - 11
Purification, cDNA cloning, and regulation of lysophospholipase from rat liver; Sugimoto H et al.; A lysophospholipase was purified 506-fold from rat liver supernatant . The preparation gave a single 24-kDa protein band on SDS-polyacrylamide gel electrophoresis . The enzyme hydrolyzed lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, lysophosphatidylserine, and 1-oleoyl-2-acetyl-sn-glycero-3-phosphocholine at pH 6-8 . The purified enzyme was used for the preparation of antibody and peptide sequencing . A cDNA clone was isolated by screening a rat liver lambda gt11 cDNA library with the antibody, followed by the selection of further extended clones from a lambda gt10 library . The isolated cDNA was 2,362 base pairs in length and contained an open reading frame encoding 230 amino acids with a Mr of 24,708 . The peptide sequences determined were found in the reading frame . When the cDNA was expressed in Escherichia coli cells as the beta-galactosidase fusion, lysophosphatidylcholine-hydrolyzing activity was markedly increased . The deduced amino acid sequence showed significant similarity to Pseudomonas fluorescence esterase A and Spirulina platensis esterase . The three sequences contained the GXSXG consensus at similar positions . The transcript was found in various tissues with the following order of abundance: spleen, heart, kidney, brain, lung, stomach, and testis = liver . In contrast, the enzyme protein was abundant in the following order: testis, liver, kidney, heart, stomach, lung, brain, and spleen . Thus the mRNA abundance disagreed with the level of the enzyme protein in liver, testis, and spleen . When HL-60 cells were induced to differentiate into granulocytes with dimethyl sulfoxide, the 24-kDa lysophospholipase protein increased significantly, but the mRNA abundance remained essentially unchanged . Thus a posttranscriptional control mechanism is present for the regulation of 24-kDa lysophospholipase.

Cancer Res, 1996 Mar 15, 56(6), 1346 - 51
Adenovirus-mediated gene transfer to human breast tumor cells: an approach for cancer gene therapy and bone marrow purging; Seth P et al.; To examine the potential use of adenovirus vectors in cancer gene therapy as a mechanism for purging bone marrow cells of possible breast cancer contaminants, we compared the infection efficiency of adenovirus and the transfection efficiency of plasmid DNA in the presence of adenovirus in human breast cancer and bone marrow cells . Following infection of breast cancer cells with an adenovirus expressing beta-galactosidase gene, high levels of beta-galactoside activity were observed . No beta-galactosidase activity was observed in low-density human bone marrow cells . A replication-deficient adenovirus mutant dl312 enhanced the transfection efficiency of a plasmid DNA-expressing beta-galactosidase gene into breast cancer cells, and addition of a liposome, lipofectamine, further enhanced the transfection efficiency . In contrast, human bone marrow cells treated under the same conditions expressed very low levels of transfected beta-galactosidase DNA . Transfection of cells with plasmid DNA expressing a truncated but fully active Pseudomonas exotoxin gene in the presence of dl312 and lipofectamine resulted in marked breast cancer cell killing, whereas colony-forming unit granulocyte-macrophage (CFU-GM) were relatively resistant to these treatments . A recombinant adenovirus expressing human wild-type p53 protein (AdWTp53) was also highly cytotoxic to breast tumor cells . Infection of breast cancer cells with AdWTp53 (100 plaque-forming units/cell) resulted in 100% loss of the clonogenicity of breast tumor cells . However, colony formation from CFU-GM was relatively resistant to the cytotoxic effects of AdWTp53 alone or in the presence of pULI100 plasmid and lipofectamine . On the basis of these results, it is proposed that human adenoviruses are potentially useful for cancer gene therapy and bone marrow purging.

J Biol Chem, 1996 Mar 15, 271(11), 6122 - 8
Ligand-toxin hybrids directed to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein exhibit lower toxicity than native Pseudomonas exotoxin; Zdanovsky AG et al.; Pseudomonas exotoxin (PE) binds the heavy chain of the alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP) . To understand the significance of this interaction, novel toxin-derived gene fusions were constructed with two ligands that also bind this receptor . A 39-kDa cellular protein, termed RAP, binds LRP with high affinity and often co-purifies with it . Two RAP toxins were constructed, one with PE and one with diphtheria toxin (DT) . RAP, which replaced the toxins binding domains, was combined with each of the corresponding translocating and ADP-ribosylating domains . Both RAP-toxins bound LRP with an apparent higher affinity than native PE . Despite this, RAP-PE and DT-RAP were less toxic than native PE . Apparently, RAP-toxin molecules bound and entered cells but used a pathway that afforded only low efficiency of toxin transport to the cytosol . This was evident because co-internalization with adenovirus increased the toxicity of RAP-toxins by 10-fold . We speculate that the high affinity of RAP binding may not allow the toxin's translocating and ADP-ribosylating domains to reach the cytosol but rather causes the toxin to take another pathway, possibly one that leads to lysosomes . To test this hypothesis, additional RAP-PE fusions were constructed . N-terminal or C-terminal fragments of RAP were joined to PE to produce two novel fusion proteins which were likely to have reduced affinity for LRP . Both of these shorter fusion proteins exhibited greater toxicity than full-length RAP-PE . A second ligand-toxin gene fusion was constructed between plasminogen activator inhibitor type 1 and DT . DT-plasminogen activator inhibitor type 1 formed a complex with tissue-type plasminogen activator and inhibited its proteolytic activity . However, like the RAP-toxins, this hybrid was less toxic for cells than native PE.

Biochemistry, 1996 Mar 5, 35(9), 2872 - 7
Recombinant immunotoxin containing a disulfide-stabilized Fv directed at erbB2 that does not require proteolytic activation; Kuan CT et al.; PE35/e23(dsFv)KDEL is a recombinant immunotoxin composed of a recombinant form of Pseudomonas exotoxin that does not need proteolytic activation and a disulfide-stabilized Fv fragment of the anti-erbB2 monoclonal antibody e23 . In this molecule, the variable heavy (V H) domain is inserted near the carboxyl terminus of PE at position 607 and the variable light (V L) domain is connected to the V H domain by a disulfide bond engineered into the framework region . The disulfide bond forms between cysteines introduced at position 44 of V H and position 99 of V L {Reiter et al . (1994) J . Biol . Chem . 269, 18327-18331} . In contrast to other PE-derived Fv fusion proteins, this type of recombinant toxin does not need proteolytic activation of the toxin domain . PE35/e23(dsFv)KDEL is very cytotoxic toward erbB2 antigen-expressing N87 cells (IC50 = 0.8 ng/mL) despite the fact that it binds to the erbB2 protein only 25% as well as e23(dsFv)PE38KDEL, in which the dsFv moiety is located at the amino terminus of the toxin . The lower binding affinity is probably due to interference by domain III of PE with the amino terminus of e23(V H), possibly where the antigen binding sites are located . Nevertheless, the specificity of immunotoxin is still retained, and it is very stable at 37 degrees C . Because of its small size, stability, and activity without proteolytic processing, this immunotoxin may be advantageous for tumor treatment . PE35/e23(dsFv)KDEL was also used to gain information about whether reduction of the disulfide bonds connecting V H and V L occur in the endoplasmic reticulum (ER) or in a proximal compartment . To do this, we switched the ER retention sequence KDEL from the toxin--V H subunit to the V L subunit . Our results suggest that reduction of the disulfide bond connecting the dsFv heterodimer occurs before the immunotoxin reaches the ER, where translocation to the cytosol appears to occur.

FEBS Lett, 1996 Mar 4, 381(3), 213 - 6
Structure of fuscopeptins, phytotoxic metabolites of Pseudomonas fuscovaginae; Ballio A et al.; The structure of the fuscopeptins, bioactive lipodepsipeptides produced in culture by the gramineae pathogen Pseudomonas fuscovaginae, has been determined . The combined use of FAB mass spectroscopy NMR spectroscopy and chemical and enzymatic procedures allowed one to define a peptide moiety corresponding to Z-Dhb-D-Pro-L-Leu-D-Ala-D-Ala-D-Ala-D-Ala-D-Val-Gly-D-Ala-D-Val-D-Ala-D- Val-Z-Dhb-Da-Thr-L-Ala-L-Dab-D-Dab-L-Phe with the terminal carboxyl group closing a macrocyclic ring on the hydroxyl group of the allothreonine residue . The N-terminus is in turn acylated by 3-hydroxyoctanoate in fuscopeptin A and 3-hydroxydecanoate in fuscopeptin B . Some preliminary data on the biological activity of fuscopeptins are also reported.

Appl Environ Microbiol, 1996 Mar, 62(3), 1093 - 5
Purification and partial characterization of an alkaline lipase from Pseudomonas pseudoalcaligenes F-111; Lin SF et al.; An extracellular alkaline lipase of alkalophilic Pseudomonas pseudoalcaligenes F-111 was purified to homogeneity . The apparent molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 32,000, and the isoelectric point was 7.3 . With p-nitrophenyl esters as its substrates, the enzyme shows preference for C12 acyl and C14 acyl groups . It was stable in the pH range of 6 to 10, which coincides with the optimum pH range.

Acta Chem Scand, 1996 Mar, 50(3), 284 - 8
pH and ionic strength effects on electron transfer rate constants and reduction potentials of the bacterial di-heme protein Pseudomonas stutzeri cytochrome c4; Karlsson JJ et al.; We have investigated the ionic strength (0.1-0.5 M NaCl) and pH dependence (4.0-7.5) of the electron transfer (ET) rate constants for oxidation and reduction of the bacterial di-heme protein cytochrome c4 (cyt c4; Pseudomonas stutzeri, ATCC No . 11607) by {Co(bipy)3}3+/2+ (bipy = 2,2'-bipyridine) . The kinetics is bi- or tri-phasic, and a mechanism based on cooperative ET at both hemes, slow intramolecular ET and electrostatically dominated inter-heme interaction is presently best in line with all the available data . The ionic strength and pH dependence of the rate constants and reduction potentials is weak . The rate constants mostly decrease by 0-50% in the ionic strength range 0.1-0.5 M . The macroscopic potentials decrease by < 10 mV . Three of the microscopic potentials increase by 10-25 mV, while the fourth one decreases by 50 mV, but the accuracy of the microscopic reduction potential values is low . There is no pH dependence of the rate constants in the range 6.0-7.5, but most rate constants drop to half the 6.0-7.5 value in the range 4.0-6.0, leaving the reduction potentials almost unaffected . The small effects are unexpected in view of the highly charged and strongly dipolar character, and the many hydrogen bond contacts of cyt c4 . These small effects must be related to the detailed rather the overall charge distribution of cyt c4.

Microbiology, 1996 Mar, 142 ( Pt 3), 477 - 84
SYR2, a gene necessary for syringomycin growth inhibition of Saccharomyces cerevisiae; Cliften P et al.; The Pseudomonas syringae cyclic lipodepsipeptide syringomycin inhibits the growth of Saccharomyces cerevisiae . A novel yeast gene, SYR2, was found to complement two syringomycin-resistant S . cerevisiae mutants . SYR2 was cloned, sequenced, and shown to encode a 349 amino acid protein located in the endoplasmic reticulum . SYR2 was identical to SUR2, which is involved in survival during nutritional starvation . Gene disruption or overexpression of SYR2 did not affect cell viability or ergosterol levels, but did influence cellular phospholipid levels . The findings suggest that phospholipids are important for the growth inhibitory action of syringomycin.

Zentralbl Bakteriol, 1996 Mar, 283(3), 351 - 9
Experimental melioidosis in inbred mouse strains; Veljanov D et al.; Experimental infection was induced in three inbred mouse strains (BALB/c, BDF1 hybrid and C57BL) by i . p . inoculation with Pseudomonas pseudomallei . The bacterial load in the viscera and the host response induced in different compartments (blood, peritoneal cavity and organs) were determined . Blood cell parameters and peritoneal exudative cell populations were evaluated during the infection with the aid of an automated haematology analyser Technicon H-1 . It was found that all mouse strains produced a similar intraperitoneal inflammatory response with predominance of granulocytes at the early stage of infection and subsequent increase of macrophages especially in BDF1 hybrid and BALB/c mice . The highest bacterial count found in the liver and spleen of C57BL was associated with corresponding tissue damage (purulent pneumonia, abscesses in liver, karyorrhexis of hepatocytes and meningoencephalitis) . The degree of bacterial load and histological changes found in BALB/c and BDF1 hybrid mice were lower than in C57BL mice . The results show that the variations in the infection magnitude among inbred mouse strains are host-dependent.

Pediatr Pulmonol, 1996 Mar, 21(3), 153 - 8
Improved survival in the Danish center-treated cystic fibrosis patients: results of aggressive treatment; Frederiksen B et al.; We report survival data for Danish center-treated cystic fibrosis (CF) patients, covering the period 1974-1993 and using cross-sectional cumulative survival probability based on annual age-specific mortality rates . Analyses by age and by years after diagnosis were made . No significant differences were noted in the survival probability when patients were grouped according to sex or absence/presence of meconium ileus . The annual mortality rate for 1989-1993 was 0-1.2% . Using the age-specific mortality rate for 1989-1993, we were unable to calculate the median survival probability because the curve did not fall below 50% (age up to 45 years); however, it was possible to show that the survival probability for a newborn CF child to reach his 45th birthday was 80.4%(confidence interval 76.5-84.6%) . The median age at diagnosis was 0.63 years with no sex difference . The probability of surviving 40 years after the diagnosis of CF was made was 83.3% (confidence interval 80.1-86.6%) . This is considerably higher than any other published survival probability . An early anti-Pseudomonas aeruginosa treatment regimen seemed important in achieving the observed improved survival.

Minerva Urol Nefrol, 1996 Mar, 48(1), 55 - 8
{Protozoan infection (Blastocystis hominis) concomitant with Pseudomonas sp . peritonitis in continuous ambulatory peritoneal dialysis (CAPD)}; Boccardo G et al.; Case-report of protozoal infection (Blastocystis bominis) during Pseudomonas peritonitis in male patient with intestinal diverticulosis on continuous ambulatory peritoneal dialysis (CAPD) treatment for chronic renal failure (CRF) . Microscopic morphology and cultural characteristics are summarized from current literature . Photographic images in phase contrast from fresh-observation of faeces and peritoneal fluid are reported . Although other Protozoa (e.g . Acanthamoeba free-living) have already been found in dialysis fluid, this is the first case, referred in literature, of Blastocystis bominis infection in CAPD patients . Some pathogenetic hypothesis are done involving Blastocystis bominis in peritoneal infection, especially in immunodepressed patients like dialysed ones . Although many chemotherapeutics are provided for this protozoiasis during enteritis, in our case no supplement was required except specific antibiotic therapy for Pseudomonas infection . Symbion or pathogen? Is now-a-day the question which troubles parasitologists . Systemic research of Protozoa in dialysed patients is anyhow advisable.

Curr Microbiol, 1996 Mar, 32(3), 147 - 50
Properties of beta-lactamase from Pseudomonas syringae; Coleman RH et al.; Pseudomonas syringae isolate BR2R produces tabtoxin, a beta-lactam-containing antibiotic, and the causative agent of wildfire disease of green bean (Phaseolus vulgaris) . beta-Lactamase production has been suggested as the mechanism that protects P . syringae from tabtoxin . We sought to determine whether the organism produces beta-lactamase and whether the enzyme plays a role in protection from this antibiotic . P . syringae and mutants defective in tabtoxin production and resistance produce beta-lactamase . Three distinct beta-lactamases with molecular weights of 41,000 were identified . The isoelectric points of the proteins were 6.1, 6.8, and 9.2 . The enzymes preferentially hydrolyze cephalosporin . This investigation demonstrates that the organism produces multiple beta-lactamases and describes characteristics of the proteins.

Plasmid, 1996 Mar, 35(2), 98 - 107
Molecular analysis of closely related copper- and streptomycin-resistance plasmids in Pseudomonas syringae pv . syringae; Sundin GW et al.; The genetic relationship of a group of copper (Cur) and streptomycin (Smr) resistance plasmids and their Pseudomonas syringae pv . syringae hosts was examined . Each of these plasmids contained sequences homologous to the oriV and par sequences from pOSU900, a cryptic P . syringae pv . syringae plasmid . Analysis of restriction digest patterns of plasmid DNA indicated that the plasmids could be clustered into four groups; two of the groups contained multiple members which differed by only a few fragments . An analysis of the host P . syringae genotypes using the arbitrarily primed PCR technique and genomic DNA indicated that the host strains could be placed in groups similar to those resulting from analysis of plasmid DNA . Southern hybridization analyses of plasmid DNA indicated that each Smr plasmid contained sequences homologous to probes specific for the strA-strB Smr genes and the transposase and resolvase genes from Tn5393 . All plasmids hybridized to two additional probes derived from P . syringae plasmid DNA, but none of the plasmids contained IS51 or IS801 sequences . Furthermore, Tn5393 was mobilized, presumably by transposition, between the incompatible plasmids pPSR5 and pPSR4 in P . syringae pv . syringae FF5 . The variation in molecular structure of the closely related plasmids in this study is similar to that observed with antibiotic-resistance plasmids from clinical bacteria.

Nephrol Dial Transplant, 1996 Mar, 11(3), 498 - 506
Longitudinal changes in peritoneal kinetics: the effects of peritoneal dialysis and peritonitis; Davies SJ et al.; BACKGROUND . Peritoneal infection and poor ultrafiltration continue to be the major causes of treatment failure in CAPD . The combined effects of peritonitis and the continuous exposure to dialysis fluid remain the most likely candidates affecting the peritoneum in the long term . The purpose of this study was to observe the effects of peritonitis and dialysis on longitudinal peritoneal function . METHODS . The peritoneal equilibration test (PET) was utilized to quantify longitudinal changes in low-molecular-weight solute transfer (D/P(creat)) and ultrafiltration (UF) in 233 patients treated with CAPD . Of these, 166 represented an unselected cohort (Group 1) studied prospectively from commencing treatment for up to 54 months, and 67 were selected patients (Group 2) with PET data available at commencement of the study, having been on dialysis for a minimum of 18 months . PETs were performed either 6-monthly or following peritonitis episodes . RESULTS . Data on the short-term effect of peritonitis kinetics were pooled for groups 1 and 2 . Single, isolated episodes (n = 86) had no significant effect on D/P(creat) or UF, whereas recurrences or clusters of infection (n = 70) caused increases in D/P(creat) and reductions in UF, the significance of which increased with the number of episodes . There were significant correlations between both changes in D/P(creat) and UF with the cumulative dialysate leukocyte count, regardless of infecting organism, suggesting that intensity of peritoneal inflammation is also important . Those organisms associated with greater change in peritoneal kinetics, e.g . S . aureus, Pseudomonas, also had the highest neutrophil counts . The longitudinal changes in peritoneal kinetics were analysed for patients in group 1 only . There was a highly significant increase in D/P(creat) after 6 months treatment; this increased further with time on treatment, reaching further significance at 42 and 48 months . There was an associated reduction in UF . In view of the short-term effects of peritonitis on kinetics group 1 was further subdivided into patients who were either peritonitis free or only experienced isolated infections, group 1a, and those that had multiple infection episodes, group 1b . Treatment drop-out, due to death or technical failure occurred at double the rate in group 1b, who also had significantly higher D/P(creat) and lower UF at 1, 6, 12, 18 and 24 months of treatment . Group 1a subsequently caught up, however, indicating that peritonitis is not the only factor influencing long-term changes in peritoneal kinetics . CONCLUSIONS . These data suggest that solute transfer increases and UF declines with time on peritoneal dialysis . This process is exacerbated and accelerated by peritonitis, and appears to be proportional to the degree of associated inflammation and number of infections in close proximity.

J Bacteriol, 1996 Mar, 178(5), 1283 - 8
A novel dipeptidyl aminopeptidase from Pseudomonas sp . strain WO24; Ogasawara W et al.; An activity similar to that of dipeptidyl aminopeptidase I (DAP I) which releases dipeptide from Gly-Arg-p-nitroanilide (Gly-Arg-pNA) was detected in a Pseudomonas sp . An enzyme was isolated and purified about 400-fold by a series of column chromatographies . The enzyme, named DAP BI (DAP from bacteria, type I), was revealed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing . The molecular mass was estimated to be 82 kDa by SDS-PAGE and 65 kDa by gel filtration, suggesting that the enzyme may be a monomer . The enzyme had an isoelectric point of 4.7 . It is optimally active at pH 9.0 . The Km and Vmax of the enzyme for Gly-Arg-pNA were 0.25 mM and 195 micromol/min/mg, respectively . The purified enzyme did not hydrolyze Gly-Phe-pNA, which was also a substrate for DAP I, whereas it hydrolyzed Arg-Arg-4-methoxy-beta-naphthylamide (Arg-Arg-MNA), a model substrate for DAP III . The Km and Vmax for Arg-Arg-MNA were 0.019 mM and 145 micromol/min/mg, respectively . This purified enzyme can also catalyze the removal of Asp-Arg from the N termini of angiotensins I and II . The enzyme activity was completely inhibited by Zn(II) (0.5 mM), tosyl-L-Lys-chloromethyl ketone (0.1 mM), and leupeptin (0.1 mM) and partially inhibited by Co(II) (0.5 mM) and chymostatin (0.1 mM), whereas the enzyme was not affected by general serine protease inhibitors (phenylmethylsulfonyl fluoride and diisopropylfluorophosphate) and thiol protease inhibitors . The substrate specificity, classification of catalytic site, and other enzymatic properties demonstrate that this enzyme is distinct from the previously described mammalian DAPs I and III and Saccharomyces cerevisiae DAP III . These results indicate that DAP BI may be a new type of the DAP family.

J Infect Dis, 1996 Mar, 173(3), 656 - 60
Ochrobactrum anthropi meningitis in pediatric pericardial allograft transplant recipients; Chang HJ et al.; An epidemiologic investigation was done after 3 patients contracted Ochrobactrum anthropi meningitis at one hospital in October 1994 . Neurosurgical patients with pericardial tissue implants were at greater risk of infection than other neurosurgical patients (3/14 vs . 0/566; P<.001) . Cultures of implants removed from 2 case-patients, an implant at implantation, a nonimplanted pericardial tissue, and an unwrapped but unopened bottle of Hank's balanced salt solution (HBSS) grew O . anthropi . Patient and tissue isolates had identical genotypes; the isolate from the HBSS bottle had a unique genotype . Culture samples from an unopened HBSS bottle and from pericardial tissue grew Pseudomonas stutzeri of the same genotype; however, no P . stutzeri infections were detected . The investigation documented intrinsic P . stutzeri contamination of HBSS . O . anthropi contamination of tissues occurred during processing, possibly due to extrinsic contamination of HBSS . Active surveillance is needed to detect infection in patients receiving transplanted tissues, and rigorous infection control practice are necessary during tissue harvesting and processing to ensure sterility.

J Bacteriol, 1996 Mar, 178(6), 1548 - 55
Suppression of a sensor kinase-dependent phenotype in Pseudomonas syringae by ribosomal proteins L35 and L20; Kitten T et al.; The lemA gene of Pseudomonas syringae pv . syringae encodes the sensor kinase of a bacterial two-component signal transduction system . Phenotypes that are lemA dependent in P . syringae include lesion formation on bean and production of extracellular protease and the antibiotic syringomycin . Recently, the gacA gene has been identified as encoding the response regulator of the lemA regulon . To identify additional components that interact with LemA, suppressors of a lemA mutation were sought . A locus was identified that, when present in multiple copies, restores extracellular protease production to a lemA insertion mutant of P . syringae pv . syringae . This locus was found to encode the P . syringae homologs of translation initiation factor IF3 and ribosomal proteins L20 and L35 of Escherichia coli and other bacteria . Deletion analysis and data from Western immunoblots with anti-IF3 antiserum suggest that protease restoration does not require IF3 . Deletion of both the L35 and L20 genes resulted in loss of protease restoration, whereas disruption of either gene alone increased protease restoration . Our results suggest that overexpression of either L20 or L35 is sufficient for protease restoration . It is unclear how alteration of ribosomal protein expression compensates in this instance for loss of a transcriptional activator, but a regulatory role for L20 and L35 apart from their function in the ribosome may be indicated.

Laryngoscope, 1996 Mar, 106(3 Pt 1), 338 - 40
SPECT gallium scintigraphy in malignant external otitis: initial staging and follow-up . Case reports; Stokkel MP et al.; Malignant external otitis (MEO) is a recognized entity characterized by a stubborn Pseudomonas external otitis; it has been most frequently observed in elderly diabetic patients . Early diagnosis is necessary for successful treatment but, despite a widespread inflammatory response, routine plain x-ray studies and computed tomography (CT) scanning show no abnormalities in its early stage . In this report, the clinical value of gallium 67 (Ga 67) single photon emission CT (SPECT) is studied in three patients suspected of having MEO, and the results are compared with findings on CT scan and laboratory tests . These results confirm that the high sensitivity of GA 67 SPECT in the initial recognition of MEO provides a more adequate technique than CT scan . Furthermore, Ga 67 scintigraphy appears to be highly accurate for follow-up evaluation of these patients.

Nat Med, 1996 Mar, 2(3), 350 - 3
Treatment of advanced solid tumors with immunotoxin LMB-1: an antibody linked to Pseudomonas exotoxin; Pai LH et al.; Immunotoxin LMB-1 is composed of monoclonal antibody B3 chemically linked to PE38, a genetically engineered form of Pseudomonas exotoxin . B3 recognizes a carbohydrate antigen (Le(Y)) present on many human solid tumors . LMB-1 has excellent antitumor activity in nude mice bearing Le(Y)-positive tumors . We conducted a phase I study of 38 patients with solid tumors who failed conventional therapy and whose tumors expressed the Le(Y) antigen . Objective antitumor activity was observed in 5 patients, 18 had stable disease, 15 progressed . A complete remission was observed in a patient with metastatic breast cancer to supraclavicular nodes . A greater than 75% tumor reduction and resolution of all clinical symptoms lasting for more than six months was observed in a colon cancer patient with extensive retroperitoneal and cervical metastasis . Three patients (two colon, one breast cancer) had minor responses . The maximum tolerated dose of LMB-1 is 75 microgram/kg given intravenously three times every other day . The major toxicity is vascular leak syndrome manifested by hypoalbuminemia, fluid retention, hypotension and, in one case, pulmonary edema . Although immunotoxins have been evaluated in clinical studies for more than two decades, this is the first report of antitumor activity in epithelial tumors.

J Biol Chem, 1996 Feb 23, 271(8), 4009 - 16
The broad substrate chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol dehydrogenase of Pseudomonas sp . strain P51 are linked evolutionarily to the enzymes for benzene and toluene degradation; Werlen C et al.; The chlorobenzene degradation pathway of Pseudomonas sp . strain P51 is an evolutionary novelty . The first enzymes of the pathway, the chlorobenzene dioxygenase and the cis-chlorobenzene dihydrodiol dehydrogenase, are encoded on a plasmid-located transposon Tn5280 . Chlorobenzene dioxygenase is a four-protein complex, formed by the gene products of tcbAa for the large subunit of the terminal oxygenase, tcbAb for the small subunit, tcbAc for the ferredoxin, and tcbAd for the NADH reductase . Directly downstream of tcbAd is the gene for the cis-chlorobenzene dihydrodiol dehydrogenase, tcbB . Homology comparisons indicated that these genes and gene products are most closely related to those for toluene (todC1C2BAD) and benzene degradation (bedC1C2BA and bnzABCD) and distantly to those for biphenyl, naphthalene, and benzoate degradation . Similar to the tod-encoded enzymes, chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol dehydrogenase were capable of oxidizing 1,2-dichlorobenzene, toluene, naphthalene, and biphenyl, but not benzoate, to the corresponding dihydrodiol and dihydroxy intermediates . These data strongly suggest that the chlorobenzene dioxygenase and dehydrogenase originated from a toluene or benzene degradation pathway, probably by horizontal gene transfer . This evolutionary event left its traces as short gene fragments directly outside the tcbAB coding regions.

EMBO J, 1996 Feb 15, 15(4), 925 - 34
A leucine zipper motif determines different functions in a DNA replication protein; Garcia de Viedma D et al.; RepA is the replication initiator protein of the Pseudomonas plasmid pPS10 and is also able to autoregulate its own synthesis . Here we report a genetic and functional analysis of a leucine zipper-like (LZ) motif located at the N-terminus of RepA . It is shown that the LZ motif modulates the equilibrium between monomeric and dimeric forms of the protein and that monomers of RepA interact with sequences at the origin of replication, oriV, while dimers are required for interactions of RepA at the repA promoter . Further, different residues of the LZ motif are seen to have different functional roles . Leucines at the d positions of the putative alpha-helix are relevant in the formation of RepA dimers required for transcriptional autoregulation . They also modulate other RepA-RepA interactions that result in cooperative binding of protein monomers to the origin of replication . The residues at the b/f positions of the putative helix play no relevant role in RepA-RepA interactions . These residues do not affect RepA autoregulation but do influence replication, as demonstrated by mutants that, without affecting binding to oriV, either increase the host range of the plasmid or are inactive in replication . It is proposed that residues in b/f positions play a relevant role in interactions between RepA and host replication factors.

Virology, 1996 Feb 15, 216(2), 455 - 8
Construction and analysis of a bacteriophage phi 6 gene 14 nonsense mutant; Casini G et al.; The production of a small low-abundance protein encoded by the large dsRNA genome segment of bacteriophage phi 6 in extracts of phage-infected Pseudomonas phaseolicola has been reported previously . Construction and analysis of a phage containing a nonsense mutation in this gene, designated gene 14, indicates that its product is not essential for growth in the laboratory . However, since early protein synthesis is delayed, burst sizes are reduced, and plaques are smaller than wild type, P14 is needed for optimal phage development.

BMJ, 1996 Feb 10, 312(7027), 338 - 45
Single or multiple daily doses of aminoglycosides: a meta-analysis; Barza M et al.; OBJECTIVE--To assess relative efficacy and toxicity of aminoglycosides given by single daily dose compared with multiple daily doses . DESIGN--Meta-analysis of 21 randomised trials identified through MEDLARS (1966 to January 1995) . Data were overviewed with fixed effects and random effects models and with meta-regression analysis . SUBJECTS--Total of 3091 patients with bacterial infection, most without pre-existing renal disease . INTERVENTIONS--Patients were randomized to receive aminoglycosides once daily or multiple times daily with similar total daily dose . MAIN OUTCOME MEASURES--Clinical failure of treatment, nephrotoxicity, ototoxicity, and mortality . RESULTS--Single daily dose regimen produced a non-significant decrease in risk of antibiotic failures (random effects risk ratio 0.83 (95% confidence interval 0.57 to 1.21)) . Benefit of once daily dosing was greater when the percentage of pseudomonas isolates in a trial was larger . Once daily administration reduced risk of nephrotoxicity (fixed effects risk ratio 0.74 (0.54 to 1.00)) . Similar trends were noted for patients with febrile neutropenia and for children . There was no significant difference in ototoxicity between the two dosing regimens, but the power of the pooled trials to detect a meaningful difference was low . There was no significant difference in mortality . CONCLUSIONS--Once daily administration of aminoglycosides in patients without pre-existing renal impairment is as effective as multiple daily dosing, has a lower risk of nephrotoxicity, and no greater risk of ototoxicity . Given the additional convenience and reduced cost, once daily dosing should be the preferred mode of administration.

J Mol Biol, 1996 Feb 9, 255(5), 735 - 52
Three-dimensional structures of free form and two substrate complexes of an extradiol ring-cleavage type dioxygenase, the BphC enzyme from Pseudomonas sp . strain KKS102; Senda T et al.; The crystal structure of an enzyme having polychlorinated-biphenyl degrading activity, the BphC enzyme from Pseudomonas sp . strain KKS102, has been solved as a free form at 1.8 A resolution . This is the first three-dimensional structure among the extradiol-type dioxygenases . Based on 34,387 reflections (10.0 to 1.8 A, completeness 87.8%), a current R-factor of 20.4% (with a free R-factor of 24.3%) was obtained with a model obeying standard geometry within 0.011 A in bond lengths and 1.91 degrees in bond angles . The BphC enzyme is a homo-octamer and each subunit is composed of two domains: Domain 1 (N-terminal part) and Domain 2 (C-terminal part) . Each domain contains two repetitions of a novel folding motif (the "beta alpha beta beta beta" motif) each consisting of ca 55 amino acid residues . A single Fe ion in the active site coordinates the side-chains of three amino acid residues (His145, His209 and Glu260) and two solvent molecules . The coordination geometry is that of a square pyramid . In addition to the free form of the BphC enzyme, we have solved two three-dimensional structures of the BphC enzyme complexed with its substrates, 2,3-dihydroxybiphenyl (2,3-DHBP) or 3-methylcatechol (3-MCT) . These substrates were found intact in the active site probably because of the oxidation of the Fe ion into ferric form (as judged by EPR spectra) in the present crystals . In both of the two substrate complexes, the two hydroxyl groups of the substrate, together with the three enzymatic side-chain ligands, were found to form a penta-coordinated system around the Fe ion roughly arranged in a trigonal bipyramidal configuration . The active site structures appear to be essentially consistent with the reaction mechanism proposed so far.

Int J Cancer, 1996 Feb 8, 65(4), 538 - 46
A bivalent single-chain antibody-toxin specific for ErbB-2 and the EGF receptor; Schmidt M et al.; ErbB-2 and EGF receptors are often co-expressed in human tumors and have been shown to synergize in the transformation of cells in experimental model systems . Transactivation of ErbB-2 can occur via ligand-induced heterodimerization with EGF receptor or other members of the ErbB family of receptor tyrosine kinases . We have previously described the potent anti-tumoral activity of the monospecific single-chain antibody-toxins scFv(FRP5)-ETA and scFv(225)-ETA binding to, respectively, ErbB-2 and the EGF receptor . Here we report the construction and functional characterization of a novel bivalent, bispecific single-chain antibody-toxin, scFv2(FRP5/225)-ETA . The fusion protein consists of 2 scFv domains specific for ErbB-2 and the EGF receptor linked to a modified Pseudomonas exotoxin A . ScFv2(FRP5/225)-ETA displayed in vitro cell killing activity on tumor cells overexpressing either ErbB-2 or the EGF receptor similar to that of the monospecific toxins . It was more potent in vitro and in vivo in inhibiting the growth of tumor cells expressing both receptors . Treatment of A431 cells with scFv2(FRP5/225)-ETA led to an increase in EGF receptor and ErbB-2 phosphotyrosine content, most likely via the induction of receptor heterodimers . This may explain the enhanced toxicity of the bispecific antibody-toxin.

Proc Natl Acad Sci U S A, 1996 Feb 6, 93(3), 974 - 8
Improved antitumor activity of a recombinant anti-Lewis(y) immunotoxin not requiring proteolytic activation; Kuan CT et al.; B1(dsFv)-PE33 is a recombinant immunotoxin composed of a mutant form of Pseudomonas exotoxin (PE) that does not need proteolytic activation and a disulfide-stabilized Fv fragment of the anti-Lewis(y) monoclonal antibody B1, which recognizes a carbohydrate epitope on human carcinoma cells . In this molecule, amino acids 1-279 of PE are deleted and domain Ib (amino acids 365-394) is replaced by the heavy chain variable region (VH) domain of monoclonal antibody B1 . The light chain (VL) domain is connected to the VH domain by a disulfide bond . This recombinant toxin, termed B1(dsFv)-PE33, does not require proteolytic activation and it is smaller than other immunotoxins directed at Lewis(y), all of which require proteolytic activation . Furthermore, it is more cytotoxic to antigen-positive cell lines . B1(dsFv)-PE38 has the highest antitumor activity of anti-Lewis(y) immunotoxins previously constructed . B1(dsFv)-PE33 caused complete regression of tumors when given at 12 micrograms/kg (200 pmol/kg) every other day for three doses, whereas B1(dsFv)-PE38 did not cause regressions at 13 micrograms/kg (200 pmol/kg) . By bypassing the need for proteolytic activation and decreasing molecular size we have enlarged the therapeutic window for the treatment of human cancers growing in mice, so that complete remissions are observed at 2.5% of the LD50.

Occup Environ Med, 1996 Feb, 53(2), 106 - 11
Sump bay fever: inhalational fever associated with a biologically contaminated water aerosol; Anderson K et al.; OBJECTIVE: To investigate the clinical, serological, and environmental features of a work related inhalational fever associated with exposure to an aerosol generated from a biologically contaminated 130,000 gallon water pool in a building used for testing scientific equipment . METHOD: Cross sectional survey of all exposed subjects (n = 83) by symptom questionnaire, clinical examination, spirometry, and serology for antibody to Pseudomonads, pool water extract, and endotoxin . In symptomatic patients diffusion capacity was measured, and chest radiology was performed if this was abnormal . Serial peak flow was recorded in those subjects with wheeze . Bacterial and fungal air sampling was performed before and during operation of the water pool pump mechanism . Endotoxin was measured in the trapped waters and in the pumps . Serum cotinine was measured as an objective indicator of smoking . RESULTS: Of the 20 symptomatic subjects, fever was most common in those with the highest exposure (chi 2 42.7, P < 0.001) in the sump bay when the water was (torrentially) recirculated by the water pumps . Symptoms occurred late in the working day only on days when the water pumps were used, and were independent of the serum cotinine . Pulmonary function was normal in most subjects (spirometry was normal in 79/83, diffusion capacity was low in five subjects, chest radiology was normal) . Peak flow recording did not suggest a work relation . The bacterial content of the aerosol rose from 6 to > 10,000 colony forming units per cubic metre (cfu/m3) (predominantly environmental Pseudomonads) when the pumps were operating . High endotoxin concentrations were measured in the waters and oil sumps in the pumps . Low concentrations of antibody to the organisms isolated were detected (apart from two subjects with high antibody) but there was no relation to exposure or the presence of symptoms and similar antibody was found in the serum samples from a non-exposed population . The fever symptoms settled completely with the simple expedient of changing the water and cleaning the pumps . CONCLUSION: Given the results of our study, the development of inhalational fever in this unique environment and clearly restricted cohort was closely related to the degree of exposure to contaminated aerosol and mainly occurred in the absence of distinct serological abnormality and independent of cigarette smoking.

Plant Cell, 1996 Feb, 8(2), 241 - 9
Isolation of Arabidopsis genes that differentiate between resistance responses mediated by the RPS2 and RPM1 disease resistance genes; Reuber TL et al.; The Arabidopsis disease resistance gene RPS2 is involved in recognition of bacterial pathogens carrying the avirulence gene avrRpt2, and the RPM1 resistance gene is involved in recognition of pathogens carrying avrRpm1 or avrB . We identified and cloned two Arabidopsis genes, AIG1 and AIG2 (for avrRpt2-induced gene), that exhibit RPS2- and avrRpt2-dependent induction early after infection with Pseudomonas syringae pv maculicola strain ES4326 carrying avrRpt2 . However, ES4326 carrying avrRpm1 or avrB did not induce early expression of AIG1 and AIG2 . Conversely, ES4326 carrying avrRpm1 or avrB induced early expression of the previously isolated defense-related gene ELI3, whereas ES4326 carrying avrRpt2 did not . The induction patterns of the AIG genes and ELI3 demonstrate that different resistance gene-avr gene combinations can elicit distinct defense responses . Furthermore, by examining the expression of AIG1 and ELI3 in plants infiltrated with a mixed inoculum of ES4326 carrying avrRpt2 and ES4326 carrying avrRpm1, we found that there is interference between the RPS2- and RPM1-mediated resistance responses.

Arch Biochem Biophys, 1996 Feb 1, 326(1), 39 - 47
Low-density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor on murine peritoneal macrophages mediates the binding and catabolism of low-density lipoprotein; Wu SM et al.; Low-density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor is a member of the low-density lipoprotein receptor family . It is known to bind a wide variety of unrelated ligands including alpha 2-macroglobulin-proteinase complexes, tissue plasminogen activator, apolipoprotein E-enriched very low density lipoprotein, lipoprotein lipase, and Pseudomonas exotoxin A . Receptor-associated protein (RAP), a protein which copurifies with LRP, can inhibit the binding and internalization of all known ligands to LRP . Recent studies have shown that some ligands can bind to more than one receptor in this family . However, the ability of low-density lipoprotein (LDL) to bind to LRP in addition to the LDL receptor has not been demonstrated consistently . In this study we demonstrate that LDL binds with high affinity to macrophage cell surface receptors at 4 degrees C (Kd = 1.8 nM) and competes for the binding of a receptor-recognized form of alpha 2-macroglobulin (alpha 2M*) (Ki = 3 nM) . alpha 2M* and RAP can inhibit the binding of LDL to macrophages completely (96 and 100% inhibition, respectively), after cell surface heparin has been removed by treatment with heparinase . Using a solid-phase assay, we show that LDL binds specifically, saturably, and with high affinity to purified LRP (Kd = 5 nM) . LDL can also completely inhibit the binding of alpha 2M* to purified LRP . These results indicate that LDL binds directly to LRP . The ability of LDL to cross-compete with alpha 2M* for binding to LRP suggests that LDL binds to a similar or overlapping site as alpha 2M* . In addition, the ability of alpha 2M* to inhibit most of the receptor-mediated binding of LDL to macrophages suggests that LDL receptors on murine peritoneal macrophages are predominantly LRP.

Infect Immun, 1996 Feb, 64(2), 524 - 7
Furin regulates both the activation of Pseudomonas exotoxin A and the Quantity of the toxin receptor expressed on target cells; Gu M et al.; Pseudomonas exotoxin A (PE) binds and enters mammalian cells via the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP) . The toxin then requires proteolytic cleavage to generate an enzymatically active fragment with translocates to the cell cytosol and inhibits protein synthesis . To assess the role of furin in determining toxin susceptibility, CHO cells were transfected with a mouse furin gene (CHO+fur cells) and maintained under neomycin selection . Cells expressing the transfected gene were about two- to threefold more sensitive to PE than were cells expressing only a neomycin resistance gene (CHO+neo cells) . Possible reasons for the increased toxin sensitivity include the cleavage of a greater number of PE molecules and/or the conversion of more single-chain LRP to the processed, two-chain form . Processing of LRP appears to be necessary to allow the surface display of this receptor . Results of ligand binding studies indicated that the CHO+fur cells displayed about twofold more surface-expressed LRP than did CHO+neo cells . In addition, the in vitro cleavage of PE by recombinant furin enhanced toxin potency about threefold for CHO+neo cells but enhanced it very little for CHO+fur cells . This suggested that CHO+fur cells were processing PE at close to the maximum usable rate . Together these findings suggest that furin is involved in at least two separate protein processing pathways that each contribute to the sensitivity of cells to PE.

Biochem Biophys Res Commun, 1996 Jan 5, 218(1), 17 - 21
Physical structure and expression of alkBA encoding alkane hydroxylase and rubredoxin reductase from Pseudomonas maltophilia; Lee NR et al.; The structural genes of the Pseudomonas maltophilia alk system, which are localized on the OCT plasmid were cloned as a 4.2-kilobase pair Hind III fragment . This fragment contains sequences for alkane hydroxylase gene (alkB) and rubredoxin reductase gene (alkA), respectively . The alkB gene encodes a 373-amino acid polypeptide (47.4 kD) that can be expressed at high levels in Pseudomonas and Escherichia coli . The alkBA genes were complemented with alkane hydroxylation in both bacteria . This result shows that alkBA gene is essential for alkane hydroxylation since chromosomal loci have been encoded for other enzymes involved in fatty acid oxidation.

Chin J Biotechnol, 1996, 12(2), 81 - 7
Preparation, cloning, and high level expression in E . coli of interleukin 2-pseudomonas exotoxin fusion genes; Gao J et al.; Using PCR and oligonucleotide-directed mutagenesis, recombinant plasmids which express IL2-PE fusion genes, e.g., IL2-PE40, IL2-PE40 KDEL, IL2-PE66(4Glu), and IL2-PE66(4Glu) KDEL, were constructed and expressed in E . coli at a high level . The fusion proteins were 20-30% of the total soluble bacterial proteins, existing as inclusion bodies in the host cell . In addition, there are single EcoRI, PstI, and SmaI restriction sites at the 5', 3' terminals and the linking region of the fusion genes, respectively, in the constructed expression plasmids so that these vectors could be conveniently converted to other expression vectors containing other cytokines or toxins.

Rev Latinoam Microbiol, 1996 Jan-Mar, 38(1), 45 - 64
{Metal resistance systems in Pseudomonas}; Cervantes C et al.; Several chromosome- and plasmid-encoded metal resistance genetic systems have been studied in Pseudomonas and related bacteria . Some systems are known with molecular detail whereas others are still poorly understood . The former include resistance genes for cations derived from mercury, cadmium and copper and anions from arsenic and chromium . Except for mercury, where a redox transformation occurs, extrusion of the toxic ions from the bacterial cytoplasm appears to be the most common mechanism of resistance.

Chirality, 1996, 8(6), 418 - 22
Kinetic resolution of amino acid esters catalyzed by lipases; Houng JY et al.; Racemic amino acids were resolved by lipase via hydrolysis of their esters . Lipases (Pseudomonas lipase from Amano PS, Rhizopus lipase from Serva, and porcine pancrease lipase from Sigma) could selectively hydrolyze the L-amino acid esters in aqueous solution with high reactivities and selectivities . The effect of the structural changes in the ester moiety on the stereoselectivity of the lipases was also investigated using D,L-homophenylalanine as a model . Procedures were developed for the resolution of natural and unnatural amino acids.

Breast Cancer Res Treat, 1996, 38(1), 3 - 9
Recombinant immunotoxins; Pastan I et al.; Pseudomonas exotoxin has been genetically modified so that it targets cancer cells . This was accomplished by deleting its cell binding domain and replacing it with Fv fragments of antibodies that react with breast, colon, and other cancers . Several recombinant immunotoxins are now in clinical trials.

Zh Mikrobiol Epidemiol Immunobiol, 1996 Jan-Feb, (1), 62 - 5
{The immune response to Pseudomonas pseudomallei in vitro}; Titova NG et al.; The results of studies made with the aim of selecting conditions for inducing immune response to P.pseudomallei are presented . The study revealed that the dose of the antigen, the amount of macrophages and the development of the process in stages are the decisive factors for inducing immune response . The proliferation of macrophages till their predominance in the culture was found to depend on the dose of the antigen . Some mitogens increased the antigen-induced proliferation of macrophages . The intensive in vitro synthesis of specific antibodies was observed at minimal doses of the antigen and the moderate content of macrophages . Under the action of mitogens and large doses of the antigen which induced the proliferation macrophages antibody synthesis was absent.

Zh Mikrobiol Epidemiol Immunobiol, 1996 Jan-Feb, (1), 16 - 9
{Thermostable Pseudomonas pseudomallei hemolysin}; Denisov II et al.; The chemical nature of thermostable P.pseudomallei hemolysin has been determined . This substance is glycolipid, synthesized by bacteria at the stationary phase of growth under the conditions of the prevalence of the source of carbon over the source of nitrogen in the nutrient medium . The hemolytic activity of thermostable hemolysin has a delayed character and is more pronounced in P.pseudomallei R-forms.

Br J Pharmacol, 1996 Jan, 117(2), 293 - 8
Enhanced vasocontraction of rat tail arteries by toxoflavin; Wang Z et al.; 1 . It has been suggested that the toxic effect of toxoflavin (TXF) produced by Pseudomonas cocovenenas is mainly due to the impairment of electron transfer of the mitochondrial respiratory chain . However, the cardiovascular effect of TXF is unknown . In the present study, the effect of TXF on the isometric contraction of rat isolated tail artery strips and the underlying mechanisms were investigated . 2 . The basal force of the tissues was not affected by the toxin . However, the application of TXF before or during KCl (60 mM) stimulation potentiated KCl-induced vasocontraction, specifically the tonic phase of the contraction . 3 . When the vessel strips were precontracted with phenylephrine (Phe), TXF further enhanced the tonic contraction of the tissue . Pretreatment of tissues with TXF also potentiated subsequent vasocontraction induced by Phe . The vasocontractor effects of TXF and Phe, however, were not additive . 4 . The vascular effect of TXF was not mediated by oxygen-derived free radicals since catalase and SOD did not affect TXF-enhanced vasocontraction . In contrast, the vasocontractor effect of TXF was dependent on extracellular Ca2+ and abolished by nifedipine (a Ca2+ antagonist) . TXF also had no effect on caffeine- or U46619-induced vasocontraction . 5 . It is suggested that TXF may potentially contract blood vessels via its effect on Ca2+ channels . This effect of TXF depends on the contractile status of the vascular tissues.

Antibiot Khimioter, 1996 Jan, 41(1), 13 - 8
{Various indices of the infectious process in treatment of glanders in monkeys}; Manzeniuk IN et al.; The time course of the clinical, biochemical and serological indices was studied during the treatment of malleus in monkeys . The internal organs of the animals infected with Pseudomonas mallei were investigated pathomorphologically . The efficacy of the biseptol/oxalinic acid combination in the therapy of malleus was estimated.

Biosens Bioelectron, 1996, 11(5), 529 - 34
Mediated amperometric determination of ammonia with a methanol dehydrogenase from Pseudomonas sp . AM-1 immobilized carbon paste electrode; Suye S et al.; PQQ-dependent methanol dehydrogenase (EC 1.1.99.8) was immobilized onto the surface of a carbon paste electrode by the cross-linking method and the electrode was covered with a porous polycarbonate membrane . An electrocatalytic steady-state current for the oxidation of methanol was observed using this electrode in the presence of phenazine methosulphate as an electron transfer mediator and NH4Cl as an activator for enzyme activity . The electrocatalytic current at +0.2 V vs . Ag/AgCl was analyzed . The properties of the electrode were examined for the determination of ammonia . The electrode responded linearly to ammonia in the range of 3 to 60 mM . The assay took 40 to 50 s . The electrode was used repeatedly at room temperature for 15 days and retained 50% of its initial activity.

Blood Purif, 1996, 14(1), 26 - 34
Retention of cytokine-inducing substances inside high-flux dialyzers; Lufft V et al.; Reprocessing of dialyzers is often performed with nonsterile solutions possibly contaminated with bacterial-derived cytokine-inducing substances . We investigated the retention of cytokine-inducing substances inside the dialyzer during reprocessing in a closed loop in vitro hemodialysis system using a polyamide high flux membrane . After the first in vitro circulation of human whole blood, rinse of the blood compartment (BC) and reverse ultrafiltration (RUF) was performed with either cytokine-inducing substance-free saline or saline contaminated with filtrates from Pseudomonas cultures (6 ng/ml LAL-reactive material); subsequently, dialyzers were stored in 2% formaldehyde . Dialyzers were rinsed with approximately 15 liters pyrogen-free saline before the second circulation using blood from the same donor; the effluates were free of cytokine-inducing substances and formaldehyde . Before and after the blood circulations, peripheral blood mononuclear cells (PBMC) were separated and total production of IL-1 alpha and IL-1 beta was determined after overnight incubation . In noncirculated PBMC as well as in PBMC separated after whole blood circulation with pyrogen-free processed dialyzers, production of IL-1 beta was not detectable . After contaminated rinse of the BC, production of IL-1 beta could be observed (1,600 +/- 1,100 pg/ml, mean +/- SEM) . When pyrogen-free RUF was performed after contaminated BC rinse, IL-1 beta production averaged 163 +/- 92 pg/ml when using reused dialyzers, but 1,820 +/- 880 pg/ml when using new dialyzers . After reuse with pyrogen-free BC-rinse and contaminated RUF no IL-1 beta synthesis was observed; however, when pyrogen-free BC-rinse and contaminated RUF was applied to new dialyzers, IL-1 beta synthesis averaged 1,620 +/- 1,200 pg/ml . We conclude that cytokine-inducing substances are retained inside the dialyzer, probably by adsorption to the membrane as well as to the protein layer covering the membrane and are still biologically active after sterilisation . Cytokine-inducing substances adsorbed to the protein layer can be partially removed by RUF . Finally, the protein layer on the membrane appears to reduce the convective transfer of cytokine-inducing substances from the dialysate into the blood compartment.

Rev Mal Respir, 1996, 13(2), 187 - 90
{Carcinoid thymus tumor at an advanced age: diagnostic value of mediastinal needle biopsy with computerized tomography}; Perdu D et al.; Carcinoid tumour of the thymus is a rare neuroendocrine tumour particularly at an advanced age . The authors report a case of a mediastinal mass in a man aged 85, the mass had remained asymptomatic for a long time . It was decided to achieve a diagnosis because the tumour was causing local compression: a mediastinal needle biopsy under computerised tomographic control confirmed that this was a carcinoid tumour and a study of the biopsy material using an electron microscope showed neurosecretory granules . A sternotomy enabled the tumour to be excised but a post-operative Pseudomonas pneumonia led to the death of the patient . This case underlines the diagnostic place of mediastinal needle biopsy in the presence of a mediastinal tumour . The technique can be carried out under computerised tomography or ultrasonography and this can be associated with a study of the biopsy specimen using electron microscopy which enables the diagnosis to be made before any therapeutic decisions . The treatment of choice of a carcinoid tumour of the thymus is surgery which confirms the tumour limits and also its thymic origin . Tumour excision can be completed using radiotherapy or even chemotherapy.

Appl Microbiol Biotechnol, 1996 Jan, 44(5), 654 - 9
Influence of phytogenic surfactants (quillaya saponin and soya lecithin) on bio-elimination of phenanthrene and fluoranthene by three bacteria; Soeder CJ et al.; The influence of two phytogenic surfactants on the elimination of polycyclic aromatic hydrocarbons (PAH) was studied in shaken-batch cultures of three soil bacteria under axenic conditions . At sufficiently high concentrations, quillaya saponin and soybean lecithin solubilized phenanthrene or fluoranthene efficiently . However, complete solubilization of the PAH by lecithin only doubled the maximal rate of elimination of the two PAH compounds by Pseudomonas 0259, strain MKm (Rhizomonas ?) and Mycobacterium EMI 2 . By contrast, quillaya saponin did not improve PAH bioavailability, and in strain MKm it caused significant growth lags above 2.5 g/l . Simultaneously with the elimination of the PAH the bacteria utilized the surfactants as substrates for growth . Intermediate formation of PAH metabolites was noted . The results suggest that some phytogenic surfactants might improve PAH bioavailability in rhizospheres.

J Antibiot (Tokyo), 1996 Jan, 49(1), 76 - 80
A novel chitinase inhibitor from a marine bacterium, Pseudomonas sp; Izumida H et al.; A new chitinase inhibitor, CI-4, was isolated from the culture broth of a marine bacterium Pseudomonas sp . IZ208 . The structure of CI-4 was determined to be cyclo(L-Arg-D-Pro) by spectral studies and comparison with a synthetic sample . CI-4 showed inhibitory activity against chitinase.

Mol Plant Microbe Interact, 1996 Jan, 9(1), 55 - 61
Identification of a new Arabidopsis disease resistance locus, RPs4, and cloning of the corresponding avirulence gene, avrRps4, from Pseudomonas syringae pv . pisi; Hinsch M et al.; A molecular genetic approach was used to study the interaction between Arabidopsis and an avirulence gene from Pseudomonas syringae pv . pisi . P . syringae pv . pisi strain 151 induces a hypersensitive response (HR) when inoculated on the Arabidopsis accession Po-1 . A genomic cosmid library was constructed from DNA from P . syringae pv . pisi strain 151 and a cosmid was identified that causes the normally virulent P . syringae pv . tomato strain DC3000 to induce an HR on Po-1 . The cosmid was subcloned and a 1.2-kb DNA fragment conferring avirulence activity was sequenced . The single significant open reading frame within the 1.2-kb fragment was designated avrRps4 . An avrRps4-specific probe hybridizes to DNA from all P . syringae pv . pisi strains tested . P . syringae strains carrying avrRps4 induces an HR on specific accessions of both Arabidopsis and soybean . Arabidopsis accession Ws-0 is resistant to DC3000(avrRps4), whereas accession RLD is susceptible . Resistance segregates as a single dominant locus in a genetic cross between Ws-0 and RLD . This disease resistance locus, RPS4, was mapped to chromosome 5 between the molecular markers sAT2105 and KG-8.

World J Surg, 1996 Jan, 20(1), 22 - 6
Value of bronchoscopic pneumonia diagnosis: prospective study; Prokop A et al.; In a prospective clinical study we examined whether bronchoscopically controlled suctioning is preferable to the blind suctioning of mucus aspirates for bacterial identification of intensive care unit patients with pneumonia . Forty patients with clinical and radiologic signs of pneumonia underwent both bronchoscopically controlled and blind endotracheal lavage . Bronchoscopically controlled suctioning did not demonstrate greater sensitivity for identifying organisms than the results obtained from blind suctioning (58 organism were bronchoscopically identified, compared to 57 organisms identified by blind suctioning; p = 0.32, NS) . Arterial and mixed venous partial oxygen pressure and shunt also showed no significant differences 15 minutes before and after examination, nor did the blood pressure or pulse . The use of four of the bronchoscopes resulted in preinterventional contamination with Pseudomonas . Bronchoscopically controlled lavage shows no advantages over blind endotracheal lavage for diagnosing pneumonia . Blind suctioning with single-use sterile catheters can be done more quickly and inexpensively with fewer personnel and a lower complication rate.

Int J Syst Bacteriol, 1996 Jan, 46(1), 200 - 5
16S rRNA gene sequence analysis relative to genomovars of Pseudomonas stutzeri and proposal of Pseudomonas balearica sp . nov; Bennasar A et al.; We compared the 16S rRNA gene sequences of 14 strains of Pseudomonas stutzeri, including type strain CCUG 11256 and strain ZoBell (= ATCC 14405), which represented the seven P . stutzeri genomovars (DNA-DNA similarity groups) that have been described . Our sequence analysis revealed clusters which were highly correlated with genomovar clusters derived from DNA-DNA hybridization data . In addition, we identified signature nucleotide positions for each genomovar . We found that the 16S rRNA gene sequences of genomovar 6 strains SP1402T (T = type strain) and LS401 were different enough from the sequence of the type strain of P . stutzeri that these organisms should be placed in a new species, Pseudomonas balearica . The type strain of P . balearica is strain SP1402 (= DSM 6083).

Appl Environ Microbiol, 1996 Jan, 62(1), 262 - 5
Genetic structures of the genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase from biphenyl- and 4-chlorobiphenyl-degrading Pseudomonas sp . strain DJ-12; Kim E et al.; The pcbC and pcbD genes of Pseudomonas sp . strain DJ-12, a natural isolate degrading biphenyl and 4-chlorobiphenyl, encode the 2,3-dihydroxybiphenyl 1,2-dioxygenase and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase, respectively . The two genes were sequenced and appear to be present in the order pcbD-pcbC as an operon.

FEMS Microbiol Lett, 1995 Dec 15, 134(2-3), 299 - 307
Molecular analysis of genes encoding phenazine biosynthesis in the biological control bacterium . Pseudomonas aureofaciens 30-84; Pierson LS 3rd et al.; The DNA sequence of five contiguous open reading frames encoding enzymes for phenazine biosynthesis in the biological control bacterium . Pseudomonas aureofaciens 30-84 was determined . These open reading frames were named phzF, phzA, phzB, phzC and phzD . Protein PhzF is similar to 3-deoxy-D-arabino-heptulosonate-7-phosphate synthases of solanaceous plants . PhzA is similar to 2,3-dihydro-2,3-dihydroxybenzoate synthase (EntB) of Escherichia coli . PhzB shares similarity with both subunits of anthranilate synthase and the phzB open reading frame complemented an E . coli trpE mutant deficient in anthranilate synthase activity . Although phzC shares little similarity to known genes, its product is responsible for the conversion of phenazine-I-carboxylic acid to 2-hydroxy-phenazine-I-carboxylic acid . PhzD is similar to pyridoxamine phosphate oxidases . These results indicate that phenazine biosynthesis in P . aureofaciens shares similarities with the shikimic acid, enterochelin, and tryptophan biosynthetic pathways.

Biochem Biophys Res Commun, 1995 Dec 14, 217(2), 632 - 9
Determination of N-myristoyl peptide sequence both by MALDI TOF MASS and with an N-myristoyl cleaving enzyme (polymyxin acylase); Misumi S et al.; Polymyxin acylase isolated from Pseudomonas sp . M-6-3 was used as an N-myristoyl cleaving enzyme in order to determine a part of the N-terminal amino acid sequence of N-myristoyl proteins . The enzyme hydrolyzed a number of N-myristoyl oligopeptides at various hydrolysis rates but not N-myristoyl proteins . The oncogenic protein (N-myristoyl-pp60c-src) was isolated from human colon adenocarcinoma cell line COLO 320DM by two-dimensional polyacrylamide gel electrophoresis . The protein was digested with trypsin and the resultant tryptic N-myristoyl tetrapeptide (N-myristoyl-Gly-Ser-Asn-Lys) was purified by HPLC and the structure was determined both by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MASS) and by a gas-phase protein sequencer before or after treatment with the polymyxin acylase . The results suggest that the N-myristoyl peptide sequence derived from N-myristoyl proteins was clearly determined by the combined use of MALDI TOF MASS and the N-myristoyl cleaving enzyme.

Biochemistry, 1995 Dec 12, 34(49), 16074 - 81
Nickel is a specific inhibitor for the binding of activated alpha 2-macroglobulin to the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor; Hussain MM et al.; The low density receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2-MR) binds to several ligands involved in lipoprotein and protease clearance . The receptor-associated protein (RAP) inhibits the binding of all known ligands . We studied the inhibition by Ni2+ of the binding of different ligands to cells and to the purified LRP/alpha 2-MR . Ni2+ inhibited all of the specific binding of radiolabeled methylamine-activated alpha 2-macroglobulin (125I-alpha 2-M*) to rabbit aortic smooth muscle cells (SMC), rat hepatoma Fu5AH, and mouse fibroblast L cells . Ni2+ also inhibited the binding of trypsin-activated alpha 2-macroglobulin to SMC but did not affect the binding of RAP, Pseudomonas exotoxin A, or low-density lipoproteins . The inhibition of alpha 2-M* binding by Ni2+ was not due to its interaction with alpha 2-M* . Preincubation of SMC with Ni2+ followed by ligand binding suggested that Ni2+ binds to cell-surface molecules and inhibits the binding of alpha 2-M* but does not affect RAP binding . Most of the binding of alpha 2-M* to SMC was due to its binding to the LRP/alpha 2-MR, as opposed to the recently described signaling receptor, as demonstrated by the inhibition of this binding by the RAP . Moreover, the inhibition of alpha 2-M* binding to the LRP/alpha 2-MR by Ni2+ was demonstrated using purified receptor immobilized on microtiter plates . Two to three molecules of 63Ni2+ bound to the immobilized receptor with equal affinity but not to alpha 2-M* . The specific binding of alpha 2-M* to the immobilized receptor was inhibited in the presence of nickel.(ABSTRACT TRUNCATED AT 250 WORDS)

Southeast Asian J Trop Med Public Health, 1995 Dec, 26(4), 636 - 8
Geographical distribution of Pseudomonas pseudomallei in China; Yang S et al.; 1,366 samples of soil and water from southern China coastal provinces were examined for Pseudomonas pseudomallei . Data showed that 58 samples were positive for this bacteria, which is primarily distributed in Hainan Province and the coastal region of mainland . This paper confirmed the environmental presence of P . pseudomallei in China and showed that the distribution of this pathogen has at least reached a latitude of 25.5 degrees north.

Protein Eng, 1995 Dec, 8(12), 1323 - 31
Disulfide stabilization of antibody Fv: computer predictions and experimental evaluation; Reiter Y et al.; Using molecular modeling technology we have recently identified positions in conserved framework regions of Fvs which can be used to stabilize antibody Fvs by an interchain disulfide bond engineered in between the structurally conserved framework positions of the variable domains of heavy (VH) and light (VL) immunoglobulin chains (disulfide-stabilized Fv; dsFv) . The computer model indicated the existence of other potential sites in the framework regions that might be suitable for disulfide bond formation between VH and VL . The possibility of obtaining dsFvs using these positions is evaluated here experimentally by constructing dsFv immunotoxins in which the Fv moiety is fused to a truncated form of Pseudomonas exotoxin . We analyzed the extent of dsFv formation and the activity of the resulting dsFv immunotoxins, and compared various dsFv molecules with the scFv immunotoxin . Our results demonstrate that position H44-L105 is the only one which gives high production yields of active dsFv . All other positions gave either low yields and activity or completely failed to produce active dsFv . With one exception, the formation and activities of the dsFvs corresponded to the C alpha-C alpha distance between the VH and VL positions, with an optimal distance of 5.7 A producing the best dsFv . Distances of 6.0-6.9 A resulted in a low yield of protein that was still capable of binding antigen, whereas distances > 7.0 A resulted in molecules in which dsFv formation was not obtained.

EMBO J, 1995 Dec 1, 14(23), 5753 - 61
Cholera toxin elevates pathogen resistance and induces pathogenesis-related gene expression in tobacco; Beffa R et al.; In animals, plants and fungi, cholera toxin (CTX) can activate signalling pathways dependent on heterotrimeric GTP binding proteins (G-proteins) . We transformed tobacco plants with a chimeric gene encoding the A1 subunit of CTX regulated by a light-inducible wheat Cab-1 promoter . Tissues of transgenic plants expressing CTX showed greatly reduced susceptibility to the bacterial pathogen Pseudomonas tabaci, accumulated high levels of salicylic acid (SA) and constitutively expressed pathogenesis-related (PR) protein genes encoding PR-1 and the class II isoforms of PR-2 and PR-3 . In contrast, the class I isoforms of PR-2 and PR-3 known to be induced in tobacco by stress, by ethylene treatment and as part of the hypersensitive response to infection, were not induced and displayed normal regulation . In good agreement with these results, microinjection experiments demonstrated that CTX or GTP-gamma-S induced the expression of a PR1-GUS reporter gene but not that of a GLB-GUS reporter gene containing the promoter region of a gene encoding the class I isoform of PR-2 . Microinjection and grafting experiments strongly suggest that CTX-sensitive G-proteins are important in inducing the expression of a subset of PR genes and that these G-proteins act locally rather than systemically upstream of SA induction.

Acta Paediatr, 1995 Dec, 84(12), 1386 - 94
Prevalence, genetics and clinical presentation of chronic granulomatous disease in Sweden; Ahlin A et al.; To estimate the prevalence of chronic granulomatous disease (CGD) in Sweden, an inquiry asking for known and possible CGD cases was mailed to paediatric, internal medicine and infectious disease departments all over Sweden . The detected patients were characterized as to genetics and the clinical presentation . Twenty-one patients (belonging to 16 different families) were found, corresponding to a prevalence of approximately 1/450,000 individuals . The patients with X-linked disease, lacking a functional gp91phox protein (n = 12), comprised 57% and 43% of the patients had an autosomal recessive (AR) disease lacking p47phox (n = 7) or p67phox (n = 1), respectively . All unrelated patients with X-linked disease displayed different gene abnormalities such as point mutations predicting nonsense (n = 3), missense (n = 1) or splice site mutations (n = 2), but also a total deletion and a unique 40 base pair duplicature insertion . The patients with p47phox-deficiency showed a GT deletion at a GTGT tandem repeat, and the p67phox-deficient patient displayed a heterozygous in-frame deletion of AAG combined with a large deletion in the other allele . Three patients died during the study period, two from pseudomonas cepacia infections . Patients with X-linked disease had more frequent infections (mean of 1.7 per year), than the patients with AR inheritance (0.5 infections per year) . The most common infections were dermal abscesses (n = 111), followed by lymphadenitis (n = 82) and pneumonias (n = 73) . Inflammatory bowel disease-like symptoms, mimicking Crohn's disease of the colon, was seen in three CGD patients.

Genetics, 1995 Dec, 141(4), 1597 - 604
Soybean resistance genes specific for different Pseudomonas syringae avirulence genes are allelic, or closely linked, at the RPG1 locus; Ashfield T et al.; RPG1 and RPM1 are disease resistance genes in soybean and Arabidopsis, respectively, that confer resistance to Pseudomonas syringae strains expressing the avirulence gene avrB . RPM1 has recently been demonstrated to have a second specificity, also conferring resistance to P . syringae strains expressing avrRpm1 . Here we show that alleles, or closely linked genes, exist at the RPG1 locus in soybean that are specific for either avrB or avrRpm1 and thus can distinguish between these two avirulence genes.

Biophys J, 1995 Dec, 69(6), 2761 - 9
Electron spin-lattice relaxation of the {Cu(1.5) .. . Cu(1.5)} dinuclear copper center in nitrous oxide reductase; Pfenninger S et al.; Relaxation times have been obtained with time-domain EPR for the dinuclear mixed valence {CuA(1.5) .. . CuA(1.5){ S = 1/2 center in nitrous oxide reductase, N2OR, from Pseudomonas stutzeri, in the TN5 mutant defective in copper chromophore biosynthesis, in a synthetic mixed valence complex, and in type 1 and 2 copper complexes . Data confirmed that the intrinsic electron spin-lattice relaxation time, T1, for N2OR in the temperature range of 6-25 K is unusually short for copper centers . At best, a twofold increase of T1 from g perpendicular to g parallel was measured . Optimized fits of the saturation-recovery data were obtained using both double-exponential and stretched-exponential functions . The temperature dependence of the spin-lattice relaxation rate of mutant N2OR is about T5.0 with the stretched-exponential model or T3.3 and T3.9 for the model using the sum of two exponentials . These T1s are intrinsic to the mixed valence {CuA(1.5) .. . CuA(1.5)} center, and no interaction of the second copper center in wild-type N2OR with the {CuA(1.5) .. . CuA(1.5)} center has been observed . The T1 of the mixed valence center of N2OR is not only shorter than for monomeric square planar Cu(II) complexes, but also shorter than for a synthetic mixed valence complex, Cu2(N{CH2CH2NHCH2CH2NHCH2CH2}3N) . The short T1 is attributed to the vibrational modes of type 1 copper and/or the metal-metal interaction in {CuA(1.5) .. . CuA(1.5)}.

Appl Microbiol Biotechnol, 1995 Dec, 44(3-4), 539 - 45
Hydrophobic adsorption of aromatic compounds on polyurethane foam as a carbon source for Pseudomonas growth; Enkiri F et al.; The use of polyurethane foam appears to be efficient to extract hydrophobic pollutants from aqueous media . Their adsorption is the result of spontaneous hydrophobic interactions with the foam, The rate of adsorption is a function of the diffusion of the molecules into the foam as well as their hydrophilic/lipophilic balance . A mixture of different molecules modifies the adsorption capacities of each type of molecule on the foam, probably resulting from stacking phenomena between the molecules . The Pseudomonas species can grow in the presence of the polyurethane foam and be adsorbed on it . Moreover, a strain of Pseudomonas pseudoalcaligenes tested in this study can use adsorbed biphenyl as the sole carbon source . Polyurethane foam therefore shows favorable characteristics for being chosen as a method of concentrating aromatic compounds and optimizing the rate of degradation of these molecules by bacteria.

Appl Microbiol Biotechnol, 1995 Dec, 44(3-4), 479 - 83
Cloning of formate dehydrogenase gene from a methanol-utilizing bacterium Mycobacterium vaccae N10; Galkin A et al.; The gene of NAD(+)-dependent formate dehydrogenase (FDH) from Mycobacterium vaccae N10 was cloned into Escherichia coli by hybridization with digoxigenin-labeled DNA probes, which were prepared by amplification of the chromosomal DNA from the bacterium by the polymerase chain reaction with degenerate primers . The primers were designed on the basis of the most conserved parts of known sequences of FDH from different organisms . An open-reading frame of 1200 bp exhibited extremely high sequence similarity to the FDH gene of Pseudomonas sp . 101 . The deduced amino acid sequence of FDH from Mycobacterium vaccae N10 (McFDH) was identical to that of Pseudomonas sp . 101 (PsFDH) except for two amino acid residues: isoleucine-35 (threonine in PsFDH) and glutamate-61 (lysine in PsFDH) . The physicochemical properties of both enzymes appeared to be closely similar to each other, but the thermostability of McFDH was a little lower than that of PsFDH . To examine the role of the two amino acid residues in the thermostability of the enzymes, glutamate-61 of McFDH was replaced by glutaminyl, prolyl and lysyl residues by site-directed mutagenesis . All the mutant enzymes showed higher thermostability than the wild-type McFDH . The negative charge of glutamate-61 contributes to the stability of the wild-type enzyme being lower than that of PsFDH.

J Trop Med Hyg, 1995 Dec, 98(6), 379 - 91
Electron microscopy study of the mode of growth of Pseudomonas pseudomallei in vitro and in vivo; Vorachit M et al.; The mode of growth of Pseudomonas pseudomallei in culture media and in the lung tissue of infected humans and animals was studied using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) . In culture media, P . pseudomallei cells were seen to be entrapped in microcolonies within large amounts of intercellular fibrous material . The lung tissue of infected humans and animals showed that bacterial cells growing in lung tissue were surrounded by radially arranged fibres that constitute a very well defined glycocalyx structure . In the infected areas of the animal lung tissue, bacterial cell could be seen to have formed glycocalyx enclosed microcolonies that displaced host cell components, e.g . the nucleus of a phagocyte . The presence of bacteria in unusual locations indicated that effective phagocytosis was not occurring . The demonstration that cells of P . pseudomallei produce exopolysaccharide glycocalyces and form glycocalyx enclosed microcolonies in laboratory media and in lung tissue of infected humans and animals and the presence of bacteria in unusual locations contribute to a new understanding of the mechanism whereby this organism can cause persistent chronic infections.

Can J Microbiol, 1995 Dec, 41(12), 1147 - 52
Production and rheological properties of a succinoglycan from Pseudomonas sp . 31260 grown on wood hydrolysates; Meade MJ et al.; Pseudomonas sp . ATCC 31260 produced substantial amounts of anionic extracellular polysaccharide (EPS) from a mineral acid hydrolysate of wood, prepared using the "Tennessee Valley Authority" process . Partially purified EPS production approached 16.5 g/L (as hexadecyltrimethylammonium bromide precipitate) when the pH of the hydrolysate was initially adjusted to 7.5 and amended with 0.05% each of peptone and yeast extract . This EPS, now characterized as a succinoglycan, is composed of glucose, galactose, succinate, pyruvate, and acetate . Solutions of this EPS are pseudoplastic, and under specified conditions, are rheologically comparable with commercially available xanthan.

Appl Environ Microbiol, 1995 Dec, 61(12), 4284 - 90
Cloning and characterization of the genes for p-nitrobenzoate degradation from Pseudomonas pickettii YH105; Yabannavar AV et al.; Pseudomonas pickettii YH105 was isolated for its ability to utilize p-nitrobenzoate as the sole source of carbon, nitrogen, and energy . Degradation of p-nitrobenzoate by this strain proceeds through a reductive route as evidenced by the accumulation of ammonia in the culture medium during growth on p-nitrobenzoate . Enzyme assays and high-performance liquid chromatography (HPLC) analysis of culture supernatants indicate that p-nitrobenzoate is degraded through p-hydroxylaminobenzoate and protocatechuate . In order to clone the genes responsible for the initial steps in the catabolic pathway, a cosmid library was constructed with P . pickettii YH105 genomic DNA . The library was screened for clones capable of transforming p-nitrobenzoate to protocatechuate, using a plate assay specific for diphenolic compounds . HPLC analysis of culture supernatants confirmed that the cosmid clones did indeed produce protocatechuate from p-nitrobenzoate . Five positive cosmid clones that possessed this activity were identified . Restriction digests of the cosmid clones indicated that all of the clones had two EcoRI fragments in common (3.9 and 1.0 kb) . One of these cosmid clones, designated pGJZ1601, was chosen for further analysis . Subcloning and activity assay experiments localized the genes responsible for the conversion of p-nitrobenzoate to protocatechuate to a 1.4-kb SalI-SphI DNA fragment . Further subcloning experiments localized the gene coding for p-nitrobenzoate reductase, responsible for the first enzymatic step in the catabolic pathway, to a 0.8-kb SalI-ApaI DNA fragment . The gene for the second step in the catabolic pathway, coding for hydroxylaminolyase, was located adjacent to the gene for the p-nitrobenzoate reductase.

Gene, 1995 Dec 1, 166(1), 89 - 93
Cloning and sequencing of the membrane-bound hydrogenase-encoding genes (hupS and hupL) from Pseudomonas hydrogenovora; Ohtsuki T et al.; The membrane-bound hydrogenase (Hdg)-encoding structural genes were isolated from the hydrogen-utilizing bacterium Pseudomonas hydrogenovora (Ph) . Nucleotide sequence analysis revealed two genes, hupS and hupL . The hupS gene encoded a 363-amino-acid (aa) polypeptide (40.4 kDa) . The deduced aa sequence contained a putative 43-aa leader peptide sequence . The hupL gene started at 55 bp downstream from the stop codon of hupS and encoded a 622-aa polypeptide (69.3 kDa) . The disruption of Ph hupS resulted in the loss of Hdg activity.

Gene, 1995 Dec 1, 166(1), 83 - 7
An open reading frame in the approximately 28-kb tox-argk gene cluster encodes a polypeptide with homology to fatty acid desaturases; Hatziloukas E et al.; Part of an apparent open reading frame in the tox-argK gene cluster of Pseudomonas syringae pathovar phaseolicola (Psp) potentially encodes a polypeptide with sequence similarity to fatty acid desaturases (DES) . Escherichia coli B expressing this segment under T7 promoter control produced a 34-kDa polypeptide . The possible involvement of a DES in facilitating phaseolotoxin (Ptx) secretion at the low temperatures normally required for its synthesis and the evolutionary implications about the origin of the tox-argK gene cluster are discussed.

Gene, 1995 Dec 1, 166(1), 77 - 82
Cloning and comparison of mercury- and organomercurial-resistance determinants from a Pseudomonas stutzeri plasmid; Reniero D et al.; Plasmid pPB confers broad-spectrum mercury resistance (HgR) to a Pseudomonas stutzeri strain . Two pPB regions, separated by 25-30 kb and sharing homology with Tn501 mer (Hg detoxification) genes, were cloned separately and each was shown to carry a cluster of functional and independently regulated mer genes . One of the two gene clusters conferred resistance only to inorganic mercury, and had a structure identical to the classical model of narrow-spectrum mer operons . In the other cluster a novel merB gene, not homologous to the other known merB, but with the same function, was mapped upstream from merA, interposed between an organomercurial-responsive regulatory element and transport genes . Evidence suggests that merB and the other structural mer genes might be transcribed from two distinct promoters . The presence of two inverted repeat-like elements, identical to those of Tn5053, upstream from merR suggests that the pPB broad-spectrum-gene cluster could be part of a transposon-like element.

J Bacteriol, 1995 Dec, 177(23), 6894 - 901
Growth on octane alters the membrane lipid fatty acids of Pseudomonas oleovorans due to the induction of alkB and synthesis of octanol; Chen Q et al.; Growth of Pseudomonas oleovorans GPo1, which contains the OCT plasmid, on octane results in changes in the membrane phospholipid fatty acid composition . These changes were not found for GPo12, an OCT-plasmid-cured variant of GPo1, during growth in the presence or absence of octane, implying the involvement of OCT-plasmid-encoded functions . When recombinant strain GPo12(pGEc47) carrying the alk genes from the OCT plasmid was grown on octane, the cells showed the same changes in fatty acid composition as those found for GPo1, indicating that such changes result from induction and expression of the alk genes . This finding was corroborated by inducing GPo12(pGEc47) with dicyclopropylketone (DCPK), a gratuitous inducer of the alk genes . Further experiments showed that the increase of the mean acyl chain length of fatty acids is related to the expression of alkB, which encodes a major integral membrane protein, while the formation of trans unsaturated fatty acids mainly results from the effects of 1-octanol, an octane oxidation product.

Biochemistry, 1995 Nov 28, 34(47), 15453 - 8
An active site phenylalanine of 3-oxo-delta 5-steroid isomerase is catalytically important for proton transfer; Brothers PN et al.; 3-oxo-delta 5-steroid isomerase (KSI) from Pseudomonas testosteroni catalyzes the isomerization of a variety of 3-oxo-delta 5-steroids to their conjugated delta 4-isomers through the intermediate formation of a dienolate ion . This dienolate is formed by proton transfer from C-4 of the substrate to Asp-38, which then protonates the dienolate at C-6 . Catalysis is enhanced by electrophilic assistance (hydrogen bonding) to the 3-oxygen by Tyr-14 . We have investigated the effect of modifying phenylalanine-101 (F101), a hydrophobic residue that is located in the binding pocket of KSI . Two mutant enzymes (F101L and F101A) of KSI were prepared, and their kinetic properties were examined with 5-androstene-3, 17-dione (1) as the substrate . Both of the mutants show reduced values of kcat compared to the wild type (WT), by about 30-fold (F101L) and by 270-fold (F101A), with only a small difference in Km values . There is little change in the Ki's ( < or = 4-fold) for the product 4-androstene-3,17-dione (3), although both enzymes bind the intermediate analog d-equilenin (4) about 25-fold less tightly than does the WT . Fluorescence spectra of 4 bound to each of these enzymes suggest that 4 is ionized at the active site of WT, un-ionized at the active site of F101A and a mixture of these ionization states at the active site of F101L . Free energy profiles are constructed for each of the mutant enzymes, and these are compared to the free energy profile for the WT.(ABSTRACT TRUNCATED AT 250 WORDS)

FEMS Microbiol Lett, 1995 Nov 15, 133(3), 259 - 64
Degradation of chlorobiphenyls catalyzed by the bph-encoded biphenyl-2,3-dioxygenase and biphenyl-2,3-dihydrodiol-2,3-dehydrogenase of Pseudomonas sp . LB400; Seeger M et al.; In order to characterize the metabolites produced in vivo by biphenyl-2,3-dioxygenase and biphenyl-2,3-dihydrodiol-2,3-dehydrogenase, the first two enzymes of the (polychloro)biphenyl catabolic pathway encoded by the bph locus of Pseudomonas sp . LB400, recombinant E . coli strains expressing the respective genes were constructed . Biphenyl-2,3-dioxygenase attack on 2,2'- or 2,4'-dichlorobiphenyl was shown to give rise to virtually quantitative ortho-dechlorination of these congeners by hydroxylation at the chlorinated carbon 2 and its unsubstituted neighbour . Elimination of hydrochloric acid directly leads to 2,3-dihydroxy-chlorobiphenyls and obviates the need for biphenyl-2,3-dihydrodiol-2,3-dehydrogenase for the catabolism of such congeners.

Diagn Microbiol Infect Dis, 1995 Nov, 23(3), 77 - 83
Investigation of nosocomial respiratory infection due to Pseudomonas cepacia by arbitrarily primed polymerase chain reaction; Miyawaki H et al.; We used DNA fingerprinting by the arbitrarily primed polymerase chain reaction (AP-PCR) technique for an epidemiologic investigation of Pseudomonas cepacia nosocomial isolates obtained from patients attending our hospital . This approach was compared with conventional phenotypic typing and pulsed-field gel electrophoresis (PFGE) . The patterns of gel electrophoresis of the products of AP-PCR differed significantly according to differences in the concentration of Mg2+ and in pH . AP-PCR and PFGE was identical in their resolving power, as the two methods generated four different profiles and identified the same group of strains . The AP-PCR method constitutes an easy alternative to the well-established PFGE method.

Zhonghua Zhong Liu Za Zhi, 1995 Nov, 17(6), 458 - 60
{Results of phase III clinical trial of Pseudomonas jinanensis vaccine injection (PVI) in the treatment of malignant pleural effusion}; Wang J et al.; From April 1993 to September 1994, a prospective multicenter phase III clinical trial on PVI in the management of malignant effusion was carried out . Five hundred and nine patients, including 382 with lung cancer, 54 with breast cancer, 16 with malignant lymphoma, 57 with other malignancies complicated with pleural effusion were treated with intrapleural injections of PVI . The over-all response rate was 82.7% (421/509) . For comparison, 41 patients with cancer of the lung (n = 31), breast (n = 6) and other sites (n = 4) also complicated with pleural effusion were treated by intrapleural PDD . The overall response rate in the latter treatment group was 61.0% (25/41) . Fever and local pain were the major adverse reactions in the PVI treated patients while nausea, vomiting and myelosuppression in the PDD-treated control patients.

Mol Plant Microbe Interact, 1995 Nov-Dec, 8(6), 863 - 70
Systemic acquired resistance in Arabidopsis requires salicylic acid but not ethylene; Lawton K et al.; Systemic acquired resistance (SAR) is an inducible plant response to infection by a necrotizing pathogen . In the induced plant, SAR provides broad-spectrum protection against not only the inducing pathogen, but also against other, unrelated pathogens . Both salicylic acid (SA) and SAR-gene expression have been implicated as playing important roles in the initiation and maintenance of SAR . Here, we describe the characterization of transgenic Arabidopsis plants that express the bacterial nahG gene encoding salicylate hydroxylase, an enzyme that can metabolize SA . Strong, constitutive expression of this gene prevents pathogen-induced accumulation of SA and the activation of SAR by exogenous SA . We show that SAR in Arabidopsis can be induced by inoculation with Pseudomonas syringe pv . tomato against infection by a challenge inoculation with Peronospora parasitica . This response is abolished in transgenic, nahG-expressing Arabidopsis, but not in ethylene-insensitive mutants . These experiments support the critical role of SA in SAR and show that ethylene sensitivity is not required for SAR induction . The NahG Arabidopsis plants will be important for future studies aimed at understanding the role of SA in plant disease resistance mechanisms.

Mol Plant Microbe Interact, 1995 Nov-Dec, 8(6), 837 - 44
Complementation analyses of Pseudomonas solanacearum extracellular polysaccharide mutants and identification of genes responsive to EpsR; McWilliams R et al.; Many plant-pathogenic bacteria require extracellular polysaccharides (EPS) for successful infection . For example, mutations tht prevent EPS production in the bacterial wilt pathogen, Pseudomonas solanacearum, will reduce the ability to wilt plants . While several P . solanacearum EPS genes have been identified and characterized, a systematic analysis of EPS mutants has not previously been performed . We have screened over 12,000 transposon-tagged mutants and categorized 66EPS::lacZ mutants into nine complementation sets . Five of these are composed of previously characterized EPS structural and regulatory loci; four contain newly described EPS loci . One of the four novel complementation sets has been determined to be defective for both EPS and lipopolysaccharide production . We also used the EPS::lacZ mutants to examine the interaction between P . solanacearum EPS genes . Overexpression of the previously described regulator EpsR was found to down-regulate the expression of genes encoding production of the major acidic form EPS.

J Neurol Sci, 1995 Nov, 133(1-2), 183 - 91
Interleukin-2 Pseudomonas exotoxin chimeric protein is cytotoxic to B cell cultures derived from myasthenia gravis patients; Steinberger I et al.; IL-2-PE664Glu is a chimeric cytotoxin consisting of interleukin-2 (IL-2) fused to a mutant form of Pseudomonas exotoxin (PE664Glu) . The chimeric cytotoxin has been previously shown to be extremely toxic to both phytohaemagglutinin blasts and mixed leukocyte reaction blasts prepared from monkey and human lymphocytes . To explore the possible clinical utility of IL-2-PE664Glu for autoimmune diseases, particularly in which B cells are involved, we tested the sensitivity of B cell lines derived from myasthenia gravis patients to this chimeric cytotoxin . 65% (15 out of 23) of the tested B cell lines were sensitive to IL-2-PE664Glu mediated cytotoxicity . B cell lines from control donors as well as from patients with another autoimmune disease, multiple sclerosis, were much less sensitive to IL-2-PE664Glu cytotoxicity . Moreover, a control protein lacking the IL-2 as the targeting moiety of the chimera, had no effect toward all B cell lines tested, thus establishing its specific activity . A detailed study of the IL-2 receptor of the patients' B cells, using the PCR technique and FACS analysis, showed that the cells express mainly the beta and gamma chains and at a lower level also the alpha-chain of the IL-2 receptor . Our results suggest that IL-2-PE664Glu could be effective for selective targeted immunotherapy of myasthenia gravis patients.

Eur J Cancer, 1995 Nov, 31A(12), 2067 - 72
Cytotoxicity of TGF alpha-PE40 and correlation to expression of epidermal growth factor receptor; Berger DP et al.; TGF alpha-PE40 is a chimeric protein composed of transforming growth factor alpha (TGF alpha) linked to a modified Pseudomonas exotoxin (PE40) . We tested the in vitro cytotoxicity of TGF alpha-PE40 on 23 different solid human tumour xenografts established in nude mice and human bone marrow cells from healthy donors, utilising a modified clonogenic assay . In order to distinguish non-specific toxicity from the targeted effects of TGF alpha-PE40, epidermal growth factor receptor (EGFR) expression of the tumours studied was assessed by Northern blot, slot blot and immunohistochemistry . TGF alpha-PE40 demonstrated differential cytotoxicity on human tumour xenografts in the clonogenic assay . No toxicity on human bone marrow cells was observed . In vitro activity of TGF alpha-PE40 showed a significant correlation with the expression of EGF receptors as determined by immunohistochemistry and slot blot . Further studies will be performed in order to determine the in vivo activity of this compound in tumour-bearing nude mice.

Appl Environ Microbiol, 1995 Nov, 61(11), 3843 - 8
Characterization and transcriptional analysis of the gene cluster for coronafacic acid, the polyketide component of the phytotoxin coronatine; Liyanage H et al.; Coronafacic acid (CFA), the polyketide component of the phytotoxin coronatine (COR), is activated and coupled to coronamic acid via amide bond formation, a biosynthetic step presumably catalyzed by the CFA ligase (cfl) gene product . The COR biosynthetic gene cluster in Pseudomonas syringae pv . glycinea PG4180 is located within a 32-kb region of a 90-kb plasmid designated p4180A . In the present study, a cloned region of p4180A complemented all CFA- mutants spanning an 18.8-kb region of the COR biosynthetic cluster . The genetic evidence presented in this study indicates that cfl and the CFA biosynthetic gene cluster are encoded by a single transcript and that transcription of all of the genes in this operon is directed by the cfl promoter . The cfl promoter was localized to a 0.37-kb region upstream of the transcriptional start site by progressive subcloning in pRG960sd, a vector containing a promoterless glucuronidase gene . Transcription of the cfl/CFA operon was temperature sensitive and showed maximal glucuronidase activity at 18 degrees C . Furthermore, transcription of the cfl/CFA operon was dependent on the functional activity of a modified two-component regulatory system located within the COR biosynthetic gene cluster . Thermoregulation of the cfl/CFA operon and the coronamic acid biosynthetic gene cluster via the modified two-component regulatory system is discussed.

J Bacteriol, 1995 Nov, 177(21), 6160 - 9
A modified two-component regulatory system is involved in temperature-dependent biosynthesis of the Pseudomonas syringae phytotoxin coronatine; Ullrich M et al.; Biosynthesis of the phytotoxin coronatine (COR) in Pseudomonas syringae pv . glycinea PG4180 is regulated by temperature at the transcriptional level . A 3.4-kb DNA fragment from the COR biosynthetic gene cluster restored temperature-regulated phytotoxin production to Tn5 mutants defective in COR production . Nucleotide sequence analysis of this fragment revealed three genes, corS, corP, and corR, which encode a modified two-component regulatory system consisting of one sensor protein, CorS, and two response regulator proteins, CorP and CorR . Although only one response regulator, CorR, had a DNA-binding domain, the phosphate-receiving domains of both response regulator proteins were highly conserved . Transcriptional fusions of the corP and corR promoters to a promoterless glucuronidase gene (uidA) indicated that these two genes are expressed constitutively at 18 and 28 degrees C . In contrast, a corS::uidA fusion exhibited the temperature dependence previously observed for COR biosynthetic promoters and exhibited maximal transcriptional activity at 18 degrees C and low activity at 28 degrees C . Furthermore, glucuronidase activity for corS::uidA was decreased in corP, corR, and corS mutants relative to the levels observed for PG4180(corS::uidA) . This difference was not observed for corP::uidA and corR::uidA transcriptional fusions since expression of these fusions remained low and constitutive regardless of the genetic background . The three regulatory genes functioned in a P . syringae strain lacking the COR gene cluster to achieve temperature-dependent activation of an introduced COR biosynthetic promoter, indicating that this triad of genes is the primary control for COR biosynthesis and responsible for thermoregulation . Our data suggest that the modified two-component regulatory system described in this study might transduce and amplify a temperature signal which results in transcriptional activation of COR biosynthetic genes.

FASEB J, 1995 Nov, 9(14), 1411 - 8
Structure and mechanism of the iron-sulfur flavoprotein phthalate dioxygenase reductase; Gassner GT et al.; Transfer of electrons between pyridine nucleotides (obligatory two-electron carriers) and hemes or {2Fe-2S} centers (obligatory one-electron carriers) is an essential step mediated by flavins in respiration, photosynthesis, and many oxygenase systems . Phthalate dioxygenase reductase (PDR), a soluble iron-sulfur flavoprotein from Pseudomonas cepacia, is a convenient model for the study of this type of electron transfer . PDR is folded into three domains; the NH2-terminal FMN binding and central NAD(H) binding domains are closely related to ferredoxin-NADP+ reductase (FNR) . The COOH-terminal {2Fe-2S} domain is similar to plant ferredoxins, and can be removed by proteolysis without significantly altering the reactivity of the FNR-like domains . Kinetic studies have identified sequential steps in the reaction of PDR with NADH that involve pyridine nucleotide binding, hydride transfer to FMN, and intramolecular electron transfer from the reduced flavin to the {2Fe-2S} cluster . Crystal structures of reduced and liganded PDR correspond to some of the intermediates formed during reduction by NADH . Small structural changes that are observed in the vicinity of the cofactors upon reduction or NAD(H) binding may provide part of the reorganization energy or contribute to the gating mechanism that controls intramolecular electron transfer.

Exp Cell Res, 1995 Nov, 221(1), 1 - 10
Protective effect of cell-permeable ceramide analogs against modeccin, ricin, Pseudomonas toxin, and diphtheria toxin; Oda T et al.; We investigated the effects of various ceramide (Cer) analogs and related sphingolipids on the cytotoxicities of modeccin, ricin, Pseudomonas toxin, and diphtheria toxin in various cell lines . The most pronounced protective effect by C6Cer, a short-chain cell-permeable Cer analog, was observed in modeccin cytotoxicity in Vero, BER-40, and MDCK cells, whereas the cytotoxicity of diphtheria toxin was not affected by any of the ceramide analogs tested . C6Cer did not affect the binding and internalization of ricin and modeccin in Vero and BER-40 cells . C2Cer and C8Cer also protected against modeccin cytotoxicity, albeit less effectively than C6Cer . However, related sphingolipids including sphingosine, sphingomyelin, lactosylceramide, C18Cer (the naturally occurring ceramide), and dihydro C6Cer had no effect . A correlation was found between the ability of ceramides to inhibit bulk protein secretion and the inhibition of modeccin cytotoxicity by ceramides . Among Cer analogs tested, C6Cer, the most potent inhibitor of modeccin cytotoxicity, strongly inhibited bulk protein secretion in Vero, BER-40, and MDCK cells . PtK1 cells, which were not protected by ceramides against toxins, were resistant to ceramide-induced inhibition of bulk protein secretion . These results confirm that Cer may modulate the intracellular transport of proteins through the Golgi complex . Such Cer-sensitive processes may be involved in the intoxication of cells by plant and bacterial toxins, especially modeccin.

J Steroid Biochem Mol Biol, 1995 Nov, 55(2), 233 - 8
Cloning, sequencing and expression of Pseudomonas testosteroni gene encoding 3 alpha-hydroxysteroid dehydrogenase; Abalain JH et al.; We describe the cloning, sequencing and expression of the 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) gene of Pseudomonas testosteroni . A genomic library of P . testosteroni total DNA constructed from SauIIIA digests ligated to an lambda gt11 vector was probed with a polyclonal antibody raised against purified enzyme . Subclones derived from a recombinant phage containing a 1746 bp insert were sequenced and found to contain an open reading frame of 696 bp that corresponds to a protein of 231 amino acid residues . A search for homologous proteins was performed . No similarity was observed when comparing 3 alpha-HSD with known members of the short-chain dehydrogenase family . However a small proteic fragment (80 amino acids) shows homology with the N-terminal sequence of bacterial L7/L12 ribosomal proteins.

Eur J Immunol, 1995 Nov, 25(11), 3094 - 9
Pertussis toxin-sensitive GTP-binding proteins regulate activation-induced apoptotic cell death of human natural killer cells; Carracedo J et al.; Apoptosis of natural killer (NK) cells can be induced by non-specific physical damage (UV irradiation, heat shock) or by simultaneous ligation of the CD16 and the interleukin-2 receptor (IL-2R) molecules, but not with either anti-CD16 or IL-2 alone . Whereas blockade of GTP-binding protein (G protein)-mediated signal transduction using ADP-ribosylating bacterial toxins or the GTPase-resistant GTP analog guanosine 5'-0-(3-thiotriphosphate (GTP gamma S) does not affect non-specific induction of NK cell apoptosis, such interventions do inhibit induction of apoptosis by anti-CD16/IL-2 . The G proteins involved in the regulation of activation-induced NK apoptosis are sensitive to pertussis toxin (PTX) and to the non-specific GTP analog GTP gamma S but not to cholera toxin, Pseudomonas exotoxin A or diphtheria toxin . A pertussis toxin mutant that lacks ADP-ribosylating activity, but conserves the membrane translocating and T cell-mitogenic effects of the native molecule, fails to inhibit NK apoptosis . To exert their apoptosis-inhibitory effect, PTX and GTP gamma S must be employed before cells are activated . Later addition has no effect, suggesting the implication of G proteins in the transmission of apoptosis-inducing signals, but not in the effector stage of apoptosis . Pre-incubation with PTX or GTP gamma S does not affect the activation of NK cells by CD16 cross-linking, IL-2 stimulation- or both, as assessed by the induction of CD69 expression, protein tyrosine phosphorylation and calcium mobilization . Moreover, neither PTX nor GTP gamma S compromise the effector function of NK cells or the susceptibility of target cells to NK-mediated lysis . These data suggest apoptosis as a novel mechanism by which NK responses may be controlled in vivo, as well as an experimental and therapeutical strategy to counteract endogenous down-regulation of NK responses.

J Thorac Cardiovasc Surg, 1995 Nov, 110(5), 1415 - 22; discussion 1422-3
Combined lung and liver transplantation in patients with cystic fibrosis . A 4 1/2-year experience; Couetil JP et al.; Patients with cystic fibrosis who have end-stage respiratory failure and associated liver cirrhosis have been considered poor candidates for lung transplantation because of high morbidity and mortality resulting from hepatic insufficiency after the operation . Since April 1989, our policy has been to combine heart-lung or lung and liver transplantation in this group of patients . Between June 1990 and March 1995, among 25 patients accepted in the program for combined transplantation, nine died awaiting transplantation and 10 underwent one of the following procedures: heart-lung-liver transplantation (n = 5), en bloc double lung-liver transplantation (n = 1), sequential double lung-liver transplantation (n = 3), and bilateral lobar lung transplantation from a split left lung and reduced liver transplantation (n = 1) . There were 5 male and 5 female patients . The ages of the recipients ranged from 10 to 24 years . Mean forced expiratory volume in 1 second was 29% and mean forced vital capacity was 35% of predicted values . All patients were infected with resistant Pseudomonas, three with Pseudomonas cepaceia, and two patients had Aspergillus species in addition . All patients had severe cirrhosis with portal hypertension . Four patients had a history of esophageal variceal bleeding and two had had previous portosystemic shunts . The operation was performed as a two-stage procedure, the intrathoracic operation being completed before the abdominal stage was begun . Cardiopulmonary bypass was used in all patients because of poor clinical condition . Immunosuppression consisted of azathioprine, cyclosporine, and prednisone, as for isolated lung transplantation . There were two perioperative deaths, one caused by primary liver failure and the second by early lung dysfunction . For the first 3 months after transplantation pulmonary infection was the most common cause of morbidity . Other complications included tracheal stenosis (n = 1), bronchial stenosis (n = 1), biliary stricture (n = 2), and severe ascites (n = 3) . All were successfully treated . Obliterative bronchiolitis developed in three patients . This was stabilized with FK 506 in two patients; the other patient underwent retransplantation at 38 months but eventually died of bleeding . Actuarial survival was 70% at 1 year and remained unchanged at 3 years . Significant functional improvement was observed in all survivors . For patients who have chronic respiratory failure with advanced cirrhosis, lung transplantation combined with liver transplantation can be performed with a satisfactory outcome.

Biochim Biophys Acta, 1995 Oct 26, 1259(1), 9 - 17
Biochemical properties of cloned lipases from the Pseudomonas family; Svendsen A et al.; Three Pseudomonas lipases, representing three subfamilies, were analysed for pH optima, destabilization by EGTA and surfactants, phospholipase and cholesterolesterase side activities . All the Pseudomonas lipases tested showed alkaline pH optima . The Pseudomonas cepacia and the P . pseudoalcaligenes lipases were totally inhibited by EGTA at pH 9, and the latter was also fully inhibited at pH 7 . The lipase from P . mendocina was not inhibited by EGTA at any of the pH values tested . These findings indicate that a calcium binding site exists in some of the Pseudomonas lipases . The P . pseudoalcaligenes, P . cepacia and P . mendocina lipases were inhibited by the anionic surfactant SDS at concentrations between 0.01-0.5 mg/ml . The P . pseudoalcaligenes and P . cepacia lipases were not inhibited by the nonionic surfactant Brij35 in concentration up to 1 mg/ml, whereas the lipase from P . mendocina was inhibited at 0.1 mg/ml . The P . pseudoalcaligenes and P . cepacia lipases were found to possess high cholesterol esterase activity . P . pseudoalcaligenes lipase was further found to have high phospholipase activity . Ten Pseudomonas lipase sequences were compared by automatic sequence alignment . On the basis of sequence identity we have classified Pseudomonas lipases into five subfamilies.

Proc Natl Acad Sci U S A, 1995 Oct 24, 92(22), 10427 - 31
Cloning and characterization of a cellular apoptosis susceptibility gene, the human homologue to the yeast chromosome segregation gene CSE1; Brinkmann U et al.; We recently isolated human cDNA fragments that render MCF-7 breast cancer cells resistant to cell death caused by Pseudomonas exotoxin, Pseudomonas exotoxin-derived immunotoxins, diphtheria toxin, and tumor necrosis factor . We report here that one of these fragments is an antisense fragment of a gene homologous to the essential yeast chromosome segregation gene CSE1 . Cloning and analysis of the full-length cDNA of the human CSE1 homologue, which we name CAS for cellular apoptosis susceptibility gene, reveals a protein coding region with similar length (971 amino acids for CAS, 960 amino acids for CSE1) and 59% overall protein homology to the yeast CSE1 protein . The conservation of this gene indicates it has an important function in human cells consistent with the essential role of CSE1 in yeast . CAS is highly expressed in human tumor cell lines and in human testis and fetal liver, tissues that contain actively dividing cells . Furthermore, CAS expression increases when resting human fibroblasts are induced to proliferate and decreases when they are growth-arrested . Thus, CAS appears to play an important role in both toxin and tumor necrosis factor-mediated cell death, as well as in cell proliferation.

Gene, 1995 Oct 16, 164(1), 187 - 8
Sequence of a gene encoding periplasmic Pseudomonas syringae ankyrin; Klotz MG et al.; A gene encoding ankyrin (Ank) was isolated from a genomic library of the plant pathogen Pseudomonas syringae pathovar syringae strain 61 (Pss61) . The gene encodes an 183-amino-acid (aa) polypeptide which has homology to the 33-aa repeat domain of mammalian Ank and Ank homologs from other bacteria, animals and plants.

J Biol Chem, 1995 Oct 13, 270(41), 24370 - 4
A novel enzyme that cleaves the N-acyl linkage of ceramides in various glycosphingolipids as well as sphingomyelin to produce their lyso forms; Ito M et al.; We describe a novel enzyme that hydrolyzes the N-acyl linkage between fatty acids and sphingosine bases in ceramides of various sphingolipids . The enzyme was purified about 3000-fold with 5% recovery from the culture filtrate of a newly isolated bacterium (Pseudomonas sp . TK4) by ammonium sulfate precipitation followed by several steps of high performance liquid chromatography . The purified enzyme preparation was completely free of exoglycosidases, sphingomyelinase, and proteases, and showed a single protein band corresponding to a molecular mass of 52 kDa on SDS-polyacrylamide slab gel electrophoresis after staining with Coomassie Brilliant Blue . The enzyme shows quite wide specificity, i.e . it hydrolyzes both neutral and acidic glycosphingolipids, and simple glycosphingolipid cerebrosides to polysialogangliosides such as GQ1b . Furthermore the enzyme also hydrolyzes sphingomyelin to produce the respective lyso form . However, the enzyme shows hardly any activity on ceramides, indicating that it is completely different from the ceramidase (EC 3.5.1.23) reported previously . This enzyme, which is tentatively named sphingolipid ceramide N-deacylase, should greatly facilitate the further study of sphingolipids as well as lysosphingolipids.

J Biol Chem, 1995 Oct 6, 270(40), 23373 - 80
Identification of residues that stabilize the single-chain Fv of monoclonal antibodies B3; Benhar I et al.; B3(Fv)-PE38 is a recombinant single-chain immunotoxin in which the Fv portion of the B3 antibody in a single-chain form, which serves as the targeting moiety, is fused to PE38, a truncated form of Pseudomonas exotoxin A, which serves as the cytotoxic moiety . B3(Fv)-PE38 is specifically cytotoxic to many human cancer cell lines and is currently evaluated in a clinical trial . Monoclonal antibodies B3 (IgG1k) and B5 (IgMk) recognize related carbohydrate epitopes on human carcinoma cells . The Fv regions of these antibodies were previously cloned and expressed as the single-chain Fv-immunotoxins B3(Fv)-PE38 and B5(Fv)-PE38, respectively . The B3(Fv)-PE38 immunotoxin binds to antigen-positive cancer cells with a higher affinity than B5(Fv)-PE38 and is a more potent cytotoxic agent than B5(Fv)-PE38 . However, it is less stable and rapidly aggregates upon incubation at 37 degrees C . The VL domains of the two Fvs are very similar, differing by only three residues, the fourth and seventh Fr1 residues and the fifth CDR1 residue . The VH domains of the two Fvs vary considerably . To investigate whether any of the different VL residues may influence the stability of the B3(Fv), we constructed a chimeric immunotoxin containing the B3VH and the B5VL . This chimera had an improved stability and a higher apparent antigen binding affinity and cytotoxic activity when compared with B3(Fv)-PE38 . Site-specific mutagenesis was used to show that the VL M4L mutation has an important role in stabilizing B3(Fv), although residues VL Ser-7 and VL Ile-28 also play a role in the increased stability . When tested in an in vivo model system, the chimera containing the B3VH and the B5VL had an improved antitumor activity in a human xenograft mouse model . These studies indicate that the common use of degenerate ("family-specific") primers to clone Fv fragments may introduce destabilizing mutations.

Semin Cancer Biol, 1995 Oct, 6(5), 297 - 306
Targeting Pseudomonas exotoxin to hematologic malignancies; Kreitman RJ et al.; Malignant cells of hematopoietic origin often express a variety of different growth factor receptors and antigens on their surface, at levels much higher than normal cells . These malignant cells can be selectively targeted with Pseudomonas exotoxin (PE) derivatives directed by interleukins 2, 4 and 6, and by Fv fragments of monoclonal antibodies to interleukin 2 receptor (IL2R) subunits, CD22 and other antigens present on these cells . Anti-Tac(Fv)-PE38, a single-chain recombinant immunotoxin which targets cells bearing the IL2Ra, is furthest along in preclinical development and is being prepared for clinical testing in patients with IL2Ra-positive leukemia, lymphoma and Hodgkin's disease.

Semin Cancer Biol, 1995 Oct, 6(5), 289 - 95
Targeted therapy of carcinomas using BR96 sFv-PE40, a single-chain immunotoxin that binds to the Le(y) antigen; Siegall CB; Monoclonal antibody BR96 recognizes a Le(y)-related carbohydrate antigen expressed on a wide range of carcinomas . Immunotoxins composed of BR96 and a binding defective form of Pseudomonas exotoxin A were constructed both as chemical conjugates and as fusion proteins . While both forms of BR96 immunotoxin were equally cytotoxic to human carcinoma cell lines in vitro, the fusion protein form, BR96 sFv-PE40, was > 10-fold more active in vivo as an antitumor agent . BR96 sFv-PE40 was used to target established human tumor xenografts in both mice and in rats . The rat which displays the Le(y) antigen on the same normal tissues as humans appears to be an appropriate model for the preclinical evaluation of this immunotoxin . Complete regressions of lung, breast and bladder carcinomas were obtained in these models upon administration of well-tolerated doses of BR96 sFv-PE40 . The clinical limitations of BR96 sFv-PE40, as well as other immunotoxins, depend on the management and/or prevention of neutralizing anti-immunotoxin antibodies and the onset of toxicities, specifically vascular leak syndrome.

Inflammation, 1995 Oct, 19(5), 587 - 98
Nitric oxide and interleukin-8 as inflammatory components of cystic fibrosis; Francoeur C et al.; We examined the production of reactive nitrogen intermediates in the tracheo-bronchial tree of patients with cystic fibrosis (CF) . Examination of the soluble phase of sputa from 17 CF patients revealed the presence of high levels of NO2-/NO3- assayed by the Greiss reaction . We also examined the presence of the chemotactic cytokine interleukin-8 (IL-8) in these samples so as to assess another important inflammatory marker; high levels of IL-8 were present in the sputa of cystic fibrosis subjects . The elevated nitrite was not produced by the presence of Pseudomonas bacteria in the sputa, inasmuch as bacteria in culture released undetectable amounts of nitrite in culture media . Neutrophils from the sputa of CF patients with disease exacerbation released higher amounts of nitrite and IL-8 . Neutrophils from the sputa were also shown to spontaneously release substantial amounts of nitrite in the supernatants, and this release was partly blocked by the antagonist NG-mono-methyl-L-arginine (L-NMMA) . Blood neutrophils were shown to release nitrite only in response to challenge with CF-associated strains of Pseudomonas, and not exposure to cytokines . There was no significant differences in nitrite release between normal and CF blood polymorphonuclear leucocytes (PMNs) . A study of upper airway epithelial cell lines showed that these cells released low amounts of nitrite after infection with CF-associated strains of Pseudomonas but not after cytokine exposure . Epithelial cell lines with CF or normal phenotypes were shown to release similar quantities of nitrite, upon stimulation with Pseudomonas . These data demonstrate that elevated levels of reactive nitrogen intermediates and IL-8 are produced in the tracheo-bronchial tree of subjects with CF . Levels of IL-8 and nitrite were higher in the secretions of CF subjects with disease exacerbation . The involvement of nitric oxide and other reactive nitrogen intermediates produced by neutrophils and other cells in the tissue damaging processes in CF deserves further investigation.

Biosci Biotechnol Biochem, 1995 Oct, 59(10), 1866 - 8
Cloning of a lignostilbene-alpha,beta-dioxygenase isozyme gene from Pseudomonas paucimobilis TMY1009; Kamoda S et al.; A genomic DNA library of Pseudomonas paucimobilis TMY1009 was constructed using a cosmid vector pWE15 . Screening of the library for lignostilbene-alpha,beta-dioxygenase (LSD) isozyme genes was done with a common probe for the alpha, beta, and gamma subunits, which composed LSD isozymes . The positive clones obtained by colony hybridization were further confirmed by Southern hybridization . A 4.2-kb BamHI-HindIII fragment hybridized with the probe was subcloned into pUC118 to yield the plasmid pKHE1700 . Escherichia coli MV1184 carrying pKHE1700 produced one of the LSD isozymes . The cloned LSD was purified and compared with LSD isozymes from P . paucimobilis TMY1009 . The behavior on column chromatographies through all purification steps accorded with that of LSD-III . Furthermore, the mobility on polyacrylamide gel electrophoresis, the elution profile on reversed-phase HPLC, and the partial amino acid sequence were common to the cloned LSD and the native LSD-III . Thus the cloned LSD could be identified as LSD-III . It was found that the gene of LSD-III (lsdB) was composed of 489 codons.

J Bacteriol, 1995 Oct, 177(20), 5834 - 9
Purification and characterization of the oxygenase component of biphenyl 2,3-dioxygenase from Pseudomonas sp . strain LB400; Haddock JD et al.; The iron-sulfur protein of biphenyl 2,3-dioxygenase (ISPBPH) was purified from Pseudomonas sp . strain LB400 . The protein is composed of a 1:1 ratio of a large (alpha) subunit with an estimated molecular weight of 53,300 and a small (beta) subunit with an estimated molecular weight of 27,300 . The native molecular weight was 209,000, indicating that the protein adopts an alpha 3 beta 3 native conformation . Measurements of iron and acid-labile sulfide gave 2 mol of each per mol of alpha beta heterodimer . The absorbance spectrum showed peaks at 325 and 450 nm with a broad shoulder at 550 nm . The spectrum was bleached upon reduction of the protein with NADPH in the presence of catalytic amounts of ferredoxinBPH and ferredoxinBPH oxidoreductase . The electron paramagnetic resonance spectrum of the reduced protein showed three signals at gx = 1.74, gy = 1.92, and gz = 2.01 . These properties are characteristic of proteins that contain a Rieske-type {2Fe-2S} center . Biphenyl was oxidized to cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene by ISPBPH in the presence of ferredoxinBPH, ferredoxinBPH oxidoreductase, NADPH, and ferrous iron . Naphthalene was also oxidized to a cis-dihydrodiol, but only 3% was converted to product under the same conditions that gave 92% oxidation of biphenyl . Benzene, toluene, 2,5-dichlorotoluene, carbazole, and dibenzothiophene were not oxidized . ISPBPH is proposed to be the terminal oxygenase component of biphenyl 2,3-dioxygenase where substrate binding and oxidation occur via addition of molecular oxygen and two reducing equivalents.

Plant Cell, 1995 Oct, 7(10), 1537 - 44
Intergeneric transfer and functional expression of the tomato disease resistance gene Pto; Rommens CM et al.; Plant disease resistance loci have been used successfully in breeding programs to transfer traits from resistant germplasm to susceptible plant cultivars . The molecular cloning of plant disease resistance genes now permits the transfer of such traits across species boundaries by genetic transformation of recipient hosts . The tomato disease resistance gene Pto confers resistance to strains of the bacterial pathogen Pseudomonas syringae pv tomato expressing the avirulence gene avrPto . Transformation of Nicotiana benthamiana with Pto results in specific resistance to P . s . pv tabaci strains carrying avrPto . The resistant phenotype is manifested by a strong inhibition of bacterial growth and the ability to exhibit a hypersensitive response . Resistance cosegregates with the Pto gene in transgene selfings and testcrosses . Our results demonstrate the conservation of disease resistance functions across genus boundaries and suggest that the utility of host-specific resistance genes can be extended by intergeneric transfer.

Plant Mol Biol, 1995 Oct, 29(1), 91 - 8
Isolation of a novel Arabidopsis ozone-induced cDNA by differential display; Sharma YK et al.; We have used a reverse transcriptase polymerase chain reaction procedure (differential display) to isolate cDNAs corresponding to transcripts that accumulate in ozone-treated Arabidopsis thaliana . In this report we describe the characterization of an ozone-induced transcript, AtOZI1 . AtOZI1 mRNA in untreated plants was detected at low levels in cotyledons, leaves, and flower buds and at higher levels in roots and mature flowers . AtOZI1 mRNA accumulation was transiently induced in leaves 3- to 5-fold within the first 6 h of ozone treatment . AtOZI1 mRNA accumulation was also transiently induced 3- to 6-fold by phytopathogenic Pseudomonas strains . Sequence analysis of AtOZI1 revealed that it encodes a 8.6 kDa basic protein that contains a putative signal peptide and two potential phosphorylation sites . Our results suggest that AtOZI1 represents a novel stress-related protein that accumulates in response to the production of active oxygen species.

Appl Microbiol Biotechnol, 1995 Oct, 43(5), 946 - 51
Nutrient-enhanced survival of and phenanthrene mineralization by alginate-encapsulated and free Pseudomonas sp . UG14Lr cells in creosote-contaminated soil slurries; Weir SC et al.; The effects of nutrient amendment and alginate encapsulation on survival of and phenanthrene mineralization by the bioluminescent Pseudomonas sp . UG14Lr in creosote-contaminated soil slurries were examined . UG14Lr was inoculated into creosote-contaminated soil slurries either as a free cell suspension or encapsulated in alginate beads prepared with montmorillonite clay and skim milk . Additional treatments were free-cell-inoculated slurries amended with sterile alginate beads, free-cell-inoculated and uninoculated slurries amended with skim milk only, and uninoculated, unamended slurries . Mineralization was determined by measuring 14CO2 released from radiolabelled phenanthrene . Survival was measured by selective plating and bioluminescence . Inclusion of skim milk was found to enhance both survival of and phenanthrene mineralization by free and encapsulated UG14Lr cells.

Plast Reconstr Surg, 1995 Oct, 96(5), 1154 - 61
Lower limb salvage by microvascular free-tissue transfer in patients with homozygous sickle cell disease; Weinzweig N et al.; Chronic, excruciatingly painful ulcerations of the lower extremities in patients with homozygous sickle cell anemia (HbSS) present a frustrating clinical problem for the reconstructive surgeon . Despite adequate wound care and skin grafting, there is a dismally high incidence of recurrence . Furthermore, there is a paucity of reliable locoregional fasciocutaneous, muscle, and myocutaneous flaps in the ankle region . Free-tissue transfer has become the procedure of choice for reconstruction of the lower third of the leg . However, in sickle cell anemia, does the obligate period of flap ischemia inherent in free-tissue transfer inevitably doom a flap to failure? We present our multidisciplinary experience over 55 months with five free flaps in four homozygous sickle cell anemia patients 21 to 38 years old who had chronic nonhealing leg ulcerations . Special perioperative measures included exchange transfusion to lower hemoglobin S to below 30 percent, maintaining the hematocrit at 31 to 35 percent, intraoperative flap washout and perfusion with warm heparinized saline-dextran solution, administration of dextran and aspirin intraoperatively and postoperatively, prophylactic topical and systemic anti-Pseudomonas antibiotics, supplemental oxygen, and warm ambient room temperature . Flaps included the latissimus dorsi muscle (two patients), the temporoparietal fascia (one patient), and "split" omentum for bilateral lower limb salvage (one patient) . Successful free-tissue transfer was accomplished in all patients . One patient suffered gradual partial occlusion of the microcirculation by sickled erythrocytes following a transient hypothermic, hypotensive episode . Sufficient flap tissue survived to permit skin grafting with an excellent result . Pseudomonas infection occurred in two patients (three flaps).(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1995 Sep 29, 270(39), 23218 - 25
Mutations in the elongation factor 2 gene which confer resistance to diphtheria toxin and Pseudomonas exotoxin A . Genetic and biochemical analyses; Foley BT et al.; Both diphtheria toxin and Pseudomonas exotoxin A inhibit eukaryotic protein synthesis by ADP-ribosylating diphthamide, a posttranslationally modified histidine residue present in the elongation factor 2 (EF-2) protein . Elongation factor 2 cannot be ADP-ribosylated by the toxins unless this histidine is modified . In this report we identify three new point mutations in toxin-resistant alleles of the Chinese hamster ovary cell elongation factor 2 gene . The mutations resulted in amino acid substitutions at positions 584 (serine to glycine), 714 (isoleucine to asparagine), and 719 (glycine to aspartic acid) . All three amino acid substitutions prevented the biosynthesis of diphthamide . The amount by which the toxins reduced protein synthesis in each of these mutant cell strains suggested that all three mutations also either impaired the function of EF-2 or reduced its steady state level in the cytoplasm . Western blot analysis showed that equal amounts of EF-2 were present in each of the cell strains, indicating that the mutations impaired the catalytic function of EF-2.

Biochem Biophys Res Commun, 1995 Sep 5, 214(1), 118 - 24
Pseudomonas syringae pv . syringae phytotoxins reversibly inhibit the plasma membrane H(+)-ATPase and disrupt unilamellar liposomes; Camoni L et al.; The Pseudomonas syringae pv . syringae phytotoxins syringomycin-E and syringopeptins 22-A and 25-A reversibly and noncompetitively inhibit purified H(+)-ATPase solubilized from plasma membrane of maize roots . Moreover, they increase the passive permeability to protons in phosphatidylcholine/phosphatidylethanolamine liposomes . Both effects are more pronounced with syringopeptins than with syringomycin-E . Activity on phospholipid bilayers is detectable at phytotoxin concentrations not affecting H(+)-ATPase activity.

Bioconjug Chem, 1995 Sep-Oct, 6(5), 624 - 9
Generation of a potent chimeric toxin by replacement of domain III of Pseudomonas exotoxin with ricin A chain KDEL; Pitcher C et al.; Following cellular uptake, Pseudomonas exotoxin (PE) is cleaved by cellular protease which generates an enzymatically active C-terminal fragment (amino acids 280-613) . This 37 kD fragment translocates to the cell cytosol where it ADP-ribosylates elongation factor 2 and inhibits protein synthesis . A recombinant hybrid toxin (designated PE-RTA) in which the ADP-ribosylation domain (domain 111) was replaced by the RNA N-glycosidase domain of ricin (the A chain or RTA) has been produced in E . coli . The hybrid toxin effectively and specifically depurinated 28S ribosomal RNA, indicating that the ricin A moiety folded into its native conformation . The cytotoxicity of PE-RTA for L929 cells was approximately 100-fold less than either native PE or whole ricin . However, the addition of the tetrapeptide KDEL to the C-terminus of PE-RTA (producing PE-RTA KDEL) increased cytotoxicity to the level of the native toxins . By analogy to PE, both PE-RTA and PE-RTA KDEL would be proteolytically cleaved within PE domain II during cell entry . A single amino acid substitution, believed to disrupt an essential step in the transport of the catalytically active PE fragment to the cell cytosol (Trp281 to Ala: Zdanovsky, A.G., Chiron, M., Pastan, I., and FitzGerald, D . J . (1993) J . Biol . Chem . 268, 21791-21799), reduced the cytotoxicities of both PE and PE-RTA KDEL by approximately 100-fold . Taken together, these data show that the ricin A chain component of the hybrid toxin requires essential PE-derived sequences at both the N- and C-termini of the translocating fragment . Clearly, in the context of this fusion protein, ricin A chain cannot effect its own transfer to the cytosol.

Zhonghua Yan Ke Za Zhi, 1995 Sep, 31(5), 370 - 3
{An experimental study of corneal allograft rejection with cytotoxin IL-2-PE40}; Wu J et al.; We used a new immunosuppressive agents interleukin (IL)-2-Pseudomonas exotoxin 40 (PE40) to treat corneal allograft rejection, using the Wistar rat model of heterograft-heterotopic graft rejection . 36 rats were divided randomly into the treatment and the control groups . On 2, 5, 7, 10 and 14 days after the transplantation, the transplanted corneas were removed for pathological examinations . The peripheral blood was collected for the analysis of T cell subgroups and T lymphocyte colony forming unit (T-CFU) assay . These results indicate that IL-2-PE40 can delay the development of corneal graft rejection and significantly reduce the percentage of T-helper cells . On the other hand, IL-2-PE40 can weaken the gathering capacity of the peripheral T-cells.

J Biochem (Tokyo), 1995 Sep, 118(3), 575 - 81
Interaction of the Pseudomonas cepacia DSM3959 lipase with its chaperone, LimA; Hobson AH et al.; The lipA gene of Pseudomonas cepacia DSM3959 requires a downstream gene, limA, in oder to express lipase activity . The product of the lim gene, LimA, is a molecular chaperone required during the folding of lipase in oder for the lipase to adopt an active conformation . The lipase and LimA proteins have been shown to form a complex precipitable with either an anti-lipase or anti-LimA antibody . LimA has been shown to form a 1:1 complex with with prelipase and lipase isolated from inverted question marknatural inverted question mark P . cepacia system . The mature lipase (lacking its signal peptide) has been expressed in the presence and absence of LimA in Escherichia coli . LimA can activate mature lipase during a urea denaturation-renaturation experiment, indicating that the signal peptide is not required for the lipase to be activated by LimA . The effects of various reagents on the renaturation of lipase from 8 M urea have been examined . We propose a mechanism for the function of the LimA chaperone during the production of active extracellular lipase.

J S Afr Vet Assoc, 1995 Sep, 66(3), 172 - 6
Pseudomonas spp . associated vegetative endocarditis in two horses; Travers CW et al.; This paper describes the case histories of two Thoroughbred horses, a 2-year-old colt in training and a 7-year-old broodmare, that were presented with histories of weight loss, exercise intolerance, intermittent fever, limb oedema and anaemia . Vegetative endocarditis of the mitral and tricuspid valves was diagnosed in the colt by means of echocardiography . Pseudomonas sp . endocarditis of the mitral valve was diagnosed in the mare using echocardiography and bacterial culture . The colt had secondary congestive heart failure and was euthanased on humane grounds . Pseudomonas cepacia was isolated from the vegetative lesions following the post mortem examination . The mare was sent home and treated with gentamicin at a dosage of 3.3 mg/kg intra-muscularly twice daily for ten days . Her condition improved and she returned to stud.

Mol Microbiol, 1995 Sep, 17(6), 1153 - 66
Molecular genetics and transport analysis of the copper-resistance determinant (pco) from Escherichia coli plasmid pRJ1004; Brown NL et al.; The copper-resistance determinant (pco) of Escherichia coli plasmid pRJ1004 was cloned and sequenced . Tn1000 transposon mutagenesis identified four complementation groups, mutations in any of which eliminated copper resistance . DNA sequence analysis showed that the four complementation groups contained six open reading frames, designated pco-ABCDRS . The protein product sequences derived from the nucleotide sequence show close homology between this copper-resistance system and the cop system of a plasmid pPT23D of Pseudomonas syringae pv . tomato . The PcoR and PcoS protein sequences show homology to the family of two-component sensor/responder phosphokinase regulatory systems . A seventh reading frame (pcoE) was identified from DNA sequence data, and lies downstream of a copper-regulated promoter . Transport assays with 64Cu(II) showed that the resistant cells containing the plasmid had reduced copper accumulation during the log phase of growth, while increased accumulation had previously been observed during stationary phase . Chromosomal mutants defective in cellular copper management were obtained and characterized . In two of these mutants pco resistance was rendered totally inactive, whilst in another two mutants pco complemented the defective genes . These data indicate that plasmid-borne copper resistance in E . coli is linked with chromosomal systems for copper management.

Chin Med Sci J, 1995 Sep, 10(3), 136 - 40
Cloning and expression of the gene coding for IL-2(60)-PE40, a molecular targeted protein; Zhang M et al.; It has recently been shown that chimeric toxin composed of IL2 fused tp PE40, a mutant form of Pseudomonas Exotoxin A devoid of its native cell recognition and binding domain was cytotoxic to IL2 receptor bearing cells . We here amplified the gene IL-2 (60), which codes for the N-terminal 1-60 amino acids of human IL-2 by PCR . After that, we fused it to PE40 and the new chimeric protein IL-2(60)-PE40 was expressed in E . coli . SDS-PAGE revealed that IL-2(60)-PE40 chimeric protein accounts for more than 18% of total cell proteins . As the region IL-2 binds with its receptor was defined in the N-terminal residues 8-54 of IL-2, such fusion proteins will have the same activity with IL-2-PE40 . Following primary purification, IL-2(60)-PE40 was shown to be very toxic to IL-2 receptor-positive cells but non measurable effect on the cells lacking IL-2 receptors . Such a structure has not been reported by now . The fusion protein is useful for suppressing the immune response in cases of rejection of allografts and organ transplants and as therapeutic agents for the treatment of IL-2 receptor related diseases such as autoimmune disease, ATL (adult T-cell leukemia), et al.

Mikrobiol Z, 1995 Sep-Oct, 57(5), 30 - 9
{The characteristics of the lipid A of the lipopolysaccharides in Pseudomonas syringae strains}; Zdorovenko GM et al.; Component composition of lipid A of Pseudomonas syringae pv . syringae 218 and P-55, pv . syringae (holci) 8299, pv . phaseolicola 120a, pv . atrofaciens 2399 has been studied . The lipid A composition of all the strains studied includes 3-hydroxydecanoic fatty acid (3-OH-C10:0), 2-hydroxydodecanoic (2-OH-C12:0), 3-hydroxydodecanoic (3-OH-C12:0), dodecanoic (C12:0), hexadecanoic (C16:0), octadecanoioc (C18:0), hexadecenic (C16:1), octadecenic (C18:1) fatty acids . The carbohydrate part of the lipid A macromolecule of all strains after acid hydrolysis contains ethanolamine, phosphoethanolamine, glucosamine.

Mikrobiol Z, 1995 Sep-Oct, 57(5), 26 - 9
Characterization of lipopolysaccharides from Pseudomonas syringae (serogroup II); Iakovleva LM et al.; Lipopolysaccharides (LPS) from the strains of Pseudomonas syringae pv.syringae 90a, 435, pv . atrofaciens K-1025, pv . morsprunorum CF-4 referred by Pastushenko and Simonovich (1979) to serogroup II have been studied . The strains were shown to be heterogeneous by chemical composition of core and lipid A and structure of O-specific polysaccharide . The preparations heterogeneity in serological cross reactions were also detected . O-specific polysaccharides of the strains having similar structures were not identical in serological tests . The assumption on the lipid A role in serogrouping of P . syringae strains has been advanced.

Zh Mikrobiol Epidemiol Immunobiol, 1995 Sep-Oct, (5), 32 - 6
{Pseudomonas pseudomallei and Pseudomonas mallei--capsule-forming bacteria}; Popov SF et al.; The specific features of the submicroscopic structure of the causative agents of melioidosis and glanders, grown in culture media and at the initial stages of experimental infection in guinea pigs and golden hamsters, were studied with the use of electronic cytochemistry and immunocytochemistry . P . pseudomallei and P . mallei were found to be capsule-forming bacteria . According to the data obtained with the use of electronic cytochemistry, the basis of the capsular substance P . pseudomallei and P . mallei is formed by polysaccharide biopolymers protecting these bacteria from phagocytosis.

Arch Dermatol, 1995 Sep, 131(9), 997 - 9
Scratch and sniff . The dynamic duo; Stitt WZ et al.; Are odors diagnostic? In this age of polymerase chain reactions, in situ hybridization, and immunohistochemical staining, is there any room left for the nose in diagnosing disease? Long ago, and perhaps far away, smell was crucial to describing an illness . Infectious diseases were known by their characteristics odors--scrofula as smelling like stale beer; typhoid, like freshly baked brown bread; rubella, like plucked feathers; and diphtheria, as "sweetish." Anosmics might be banned from medical school . Perhaps we have left the descriptions behind along with these illnesses we rarely encounter today . After all, how many young physicians, residents, or medical students have ever seen a case of diphtheria or even rubella, and how many fewer have ever plucked a chicken? We have learned that pellagra (that "must appear" diagnosis in our differential by rote, but not by example, for photosensitive dermatoses) should smell like sour bread and that the exotic favus should smell "mousy" (Table 1) . What does Candida smell like--a "heavy sweetness"? Darier's disease in poor control--"organic"? Pseudomonal infections--"foul and biting"? And are not our patients with noninfected eczematous dermatitis distinct for lacking any peculiar odor, do they not actually smell "dry"? We cannot blame the abandonment of our olfactory skills on the younger generation, for how many of us could describe those odors we smell every day? Would we be able to detect a subtle change in the odor of our patient with psoriasis, a change perhaps signifying superinfection?

Biodegradation, 1995 Sep, 6(3), 223 - 7
Overexpression and feasible purification of thermostable L-2-halo acid dehalogenase of Pseudomonas sp . YL; Liu JQ et al.; The gene encoding thermostable L-2-halo acid dehalogenase of Pseudomonas sp . YL was isolated, and its overexpression system was constructed . Gene library was prepared from Sau3AI fragments of total DNA from Ps . sp . YL, pUC118 as a vector and Escherichia coli JM109 as a host . The recombinant cells resistant to bromoacetate, a germicide, were isolated and shown to produce L-2-halo acid dehalogenase . Subsequently, subcloning was carried out with pKK223-3 as a vector, and the length of DNA inserted was reduced to 1.1 kbp . One of the subclones showed very high activity, and the amount of the dehalogenase produced corresponded to about 30% of the soluble protein . From 5 g (wet weight) of cells, 105 mg of dehalogenase was efficiently purified by heat treatment and DEAE-Toyopearl chromatography . This overexpression system provides a large amount of the thermostable enzyme to enable us to study the properties, structure and application of the enzyme.

Biodegradation, 1995 Sep, 6(3), 217 - 22
Cloning and expression of a haloacid dehalogenase from Pseudomonas sp . strain 19 S; Kocabiyik S et al.; A dehalogenase gene specifying the utilization of a variety of haloacids by Pseudomonas sp . Strain 19S has been cloned and expressed in E . coli . Our cloning strategy employed specific amplification of a fragment homologous to Pseudomonas dehalogenase gene by Polymerase Chain Reaction (PCR) . The PCR amplicon successfully acted as a probe to detect the dehalogenase gene in the Southern Blot of the digested Pseudomonas total DNA . Corresponding fragments were cloned into pUC 18 vector and amplified in E . coli MV 1190 . One clone with a substantial dehalogenation activity carried a recombinant plasmid containing a 5.5 kb insert.

Biodegradation, 1995 Sep, 6(3), 203 - 12
Dehalogenation of 4-chlorobenzoate . Characterisation of 4-chlorobenzoyl-coenzyme A dehalogenase from Pseudomonas sp . CBS3; Loffler F et al.; Pseudomonas sp . CBS3 is capable of growing with 4-chlorobenzoate as sole source of carbon and energy . The removal of the chlorine of 4-chlorobenzoate is performed in the first degradation step by an enzyme system consisting of three proteins . A 4-halobenzoate-coenzyme A ligase activates 4-chlorobenzoate in a coenzyme A, ATP and Mg2+ dependent reaction to 4-chlorobenzoyl-coenzyme A . This thioester intermediate is dehalogenated by the 4-chlorobenzoyl-coenzyme A dehalogenase . Finally coenzyme A is split off by a 4-hydroxybenzoyl-CoA thioesterase to form 4-hydroxybenzoate . The involved 4-chlorobenzoyl-coenzyme A dehalogenase was purified to apparent homogeneity by a five-step purification procedure . The native enzyme had an apparent molecular mass of 120,000 and was composed of four identical polypeptide subunits of 31 kDa . The enzyme displayed an isoelectric point of 6.7 . The maximal initial rate of catalysis was achieved at pH 10 at 60 degrees C . The apparent Km value for 4-chlorobenzoyl-coenzyme A was 2.4-2.7 microM . Vmax was 1.1 x 10(-7) M sec-1 (2.2 mumol min-1 mg-1 of protein) . The NH2-terminal amino acid sequence was determined . All 4-halobenzoyl-coenzyme A thioesters, except 4-fluorobenzoyl-coenzyme A, were dehalogenated by the 4-chlorobenzoyl-CoA dehalogenase.

Mol Plant Microbe Interact, 1995 Sep-Oct, 8(5), 700 - 8
Molecular characterization of the Pseudomonas syringae pv . pisi plasmid-borne avirulence gene avrPpiB which matches the R3 resistance locus in pea; Cournoyer B et al.; An avirulence gene (designated avrPpiB) from race 3 of Pseudomonas syringae pv . pisi was cloned and sequenced . The gene corresponded to a single open reading frame of 831 nt identified by transposon mutagenesis and subcloning . This ORF encodes a predicted hydrophilic protein of 276 amino acids (MW 31,300) . It effects the expression of a resistance mechanism governed by a single genetic locus in pea . Cosegregation of resistance at the R3 locus of pea was observed towards race 3 and a transconjugant carrying the cloned avrPpiB gene according to the predicted 3:1 ratio of resistant:susceptible F2 progeny from a cross between Jade (R3 R3) and Kelvedon Wonder (rr) cultivars . DNA hybridization studies showed avrPpiB to be plasmid-borne in race 3 and suggested the presence of other alleles on one of the endogenous plasmids of races 1 and 7 . Disruption of the avrPpiB allele of race 1 and its complementation confirmed its behavior towards pea cultivars expressing the R3 locus . Homologs of avrPpiB were detected in P . syringae pv . phaseolicola, P . syringae pv . maculicola, and P . syringae pv . tomato . The presence of avrPpiB homologs in P . syringae pv . phaseolicola does not match any gene-for-gene pattern of interaction with bean cultivars.

Appl Environ Microbiol, 1995 Sep, 61(9), 3373 - 8
Cloning, characterization, and expression of a gene region from Pseudomonas sp . strain ADP involved in the dechlorination of atrazine; de Souza ML et al.; We previously identified a Pseudomonas sp . strain, ADP, which rapidly metabolized atrazine in liquid culture, agar plates, and soils (R . T . Mandelbaum, D . L . Allan, L . P . Wackett, Appl . Environ . Microbiol . 61:1451-1457, 1995) . In this study, we report the cloning and partial characterization of a gene region from Pseudomonas sp . strain ADP that encodes atrazine degradation activity . A 22-kb EcoRI genomic DNA fragment, designated pMD1, was shown to encode atrazine dechlorination activity in Escherichia coli DH5 alpha . Atrazine degradation was demonstrated by a zone-clearing assay on agar medium containing crystalline atrazine and by chromatographic methods . A gene conferring the atrazine-clearing phenotype was subsequently subcloned as a 1.9-kb AvaI fragment in pACYC184, designated pMD4, and was expressed in E . coli . This result and random Tn5 mutagenesis established that the 1.9-kb AvaI fragment was essential for atrazine dechlorination . High-pressure liquid and thin-layer chromatographic analyses were used to rigorously establish that E . coli containing pMD4 degraded atrazine and accumulated hydroxyatrazine . Hydroxyatrazine was detected only transiently in E . coli containing pMD1 . This is consistent with the idea that hydroxyatrazine is the first metabolite in atrazine degradation by Pseudomonas sp . strain ADP . A 0.6-kb ApaI-PstI fragment from pMD4, containing the putative atrazine chlorohydrolase gene, hybridized to DNA from atrazine-degrading bacteria isolated in Switzerland and Louisiana.(ABSTRACT TRUNCATED AT 250 WORDS)

EMBO J, 1995 Sep 1, 14(17), 4132 - 42
Solution structure of the single-stranded DNA binding protein of the filamentous Pseudomonas phage Pf3: similarity to other proteins binding to single-stranded nucleic acids; Folmer RH et al.; The three-dimensional structure of the homodimeric single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been determined using heteronuclear multidimensional NMR techniques and restrained molecular dynamics . NMR experiments and structure calculations have been performed on a mutant protein (Phe36 --> His) that was successfully designed to reduce the tendency of the protein to aggregate . The protein monomer is composed of a five-stranded antiparallel beta-sheet from which two beta-hairpins and a large loop protrude . The structure is compared with the single-stranded DNA binding protein encoded by the filamentous Escherichia coli phage Ff, a protein with a similar biological function and DNA binding properties, yet quite different amino acid sequence, and with the major cold shock protein of Escherichia coli, a single-stranded DNA binding protein with an entirely different sequence, biological function and binding characteristics . The amino acid sequence of the latter is highly homologous to the nucleic acid binding domain (i.e . the cold shock domain) of proteins belonging to the Y-box family . Despite their differences in amino acid sequence and function, the folds of the three proteins are remarkably similar, suggesting that this is a preferred folding pattern shared by many single-stranded DNA binding proteins.

Microbiology, 1995 Sep, 141 ( Pt 9), 2339 - 50
Glucose metabolism in 'Sphingomonas elodea': pathway engineering via construction of a glucose-6-phosphate dehydrogenase insertion mutant; Vartak NB et al.; 'Sphingomonas (formerly Pseudomonas) elodea' produces the industrially important polysaccharide gellan when grown in media containing glucose . Glucose catabolic enzymes and enzymes of central carbon metabolism were assayed in crude extracts of glucose-grown cultures of this bacterium . Based on these analyses it was concluded that glucose is converted to either gluconate or glucose 6-phosphate and that both of these products are converted to 6-phosphogluconate, a precursor for the Entner-Doudoroff (ED) and pentose phosphate pathways . Phosphoglucoisomerase (Pgi) activity was detected, but the lack of phosphofructokinase activity indicated that the Embden-Meyerhof glycolytic pathway is non-functional for glucose degradation . Thus, this bacterium utilizes glucose mainly via the ED and pentose phosphate pathways . Enzyme analyses suggested the involvement of glucose-6-phosphate dehydrogenase (Zwf) in glucose utilization and CO2 production . The zwf gene was cloned from 'S . elodea' and partially sequenced, and a null zwf mutant was constructed . This mutant exhibited no Zwf activity in in vitro assays, grew normally on glucose minimal medium and accumulated biomass (cells plus gellan) and produced CO2 at the same rates as the parental strain . Potential explanations for this finding are provided . Clones carrying the pgi gene were isolated fortuitously.

Microbiology, 1995 Sep, 141 ( Pt 9), 2223 - 33
The 4-hydroxy-2-oxovalerate aldolase and acetaldehyde dehydrogenase (acylating) encoded by the nahM and nahO genes of the naphthalene catabolic plasmid pWW60-22 provide further evidence of conservation of meta-cleavage pathway gene sequences; Platt A et al.; We report the complete nucleotide sequence and over-expression of the nahOM genes for the acetaldehyde dehydrogenase (acylating) and the 4-hydroxy-2-oxovalerate aldolase from the meta pathway operon of the naphthalene catabolic plasmid pWW60-22 from Pseudomonas sp . NCIMB9816 . Additional partial sequence analysis of adjacent DNA shows the gene order within the operon to be nahNLOMK, identical to the order found for the isofunctional genes in the meta pathway operons in the toluene/xylene pathway of TOL plasmid pWW0 and the phenol/methylphenol pathway of pVI150 . The deduced amino acid sequences of NahO and NahM were significantly homologous to the equivalent enzymes encoded by other Pseudomonas meta pathways, although both were the most divergent in each comparison . The nahOM genes were inserted downstream of the T7 promoter in the expression vector pET3a and similar constructs were also made of the isofunctional regions from pVI150 (dmpFG) and TOL plasmid pDK1 (xyIQK) . High expression of all three gene pairs was detected by enzyme assays and by SDS-PAGE.

J Biol Chem, 1995 Aug 25, 270(34), 20078 - 83
Ricin cytotoxicity is sensitive to recycling between the endoplasmic reticulum and the Golgi complex; Simpson JC et al.; Cytotoxic proteins that kill mammalian cells by catalytically inhibiting protein synthesis must enter the cytosol in order to reach their substrates . With the exception of diphtheria toxin, which enters the cytosol from acidified endosomes, the intracellular site of translocation of other toxins including ricin, Escherichia coli Shiga-like toxin-1, and Pseudomonas exotoxin A is likely to involve early compartments of the secretory pathway . We have used a molecular approach to identify the site and mechanism of toxin delivery to the cytosol by transiently expressing mutant GTPases that inhibit the assembly of biochemical complexes mediating anterograde and retrograde transport in the exocytic and endocytic pathways . The results provide evidence to suggest that receptors actively recycling between the endoplasmic reticulum and terminal Golgi compartments are essential for toxin translocation to the cytosol from the endoplasmic reticulum . The rapid kinetics of intoxication demonstrate a substantial level of bidirectional membrane flow and sorting through the early secretory pathway.

J Biotechnol, 1995 Aug 15, 42(1), 9 - 22
The expression, affinity purification and characterization of recombinant Pseudomonas exotoxin 40 (PE40) secreted from Escherichia coli; Kawooya JK et al.; Procedures have been devised for producing high yields of purified recombinant PE40, a protein important for the development of the anti-AIDS therapeutic, sCD4-PE40 . PE40 is a truncated form of the bacterial toxin, Pseudomonas exotoxin A; it lacks the cell-binding domain, but retains domains II and III that are involved in membrane translocation and inhibition of protein synthesis in eukaryotic cells . Expression vectors in Escherichia coli encoding PE40 synthesized the product as a soluble protein under control of the T7 promoter . The expression capabilities of transformants of E . coli BL21(DE3) were highly unstable . Expression levels (secreted and total) were evaluated in shake flasks and at the 10-1 scale at 27 degrees C and 37 degrees C, and following induction by IPTG or lactose . The cell-free media from the batch process was applied directly to a Cibacron blue F3GA-chromatographic medium and PE40 was eluted by nicotinamide in high yield and purity . This purification strategy was based on the structural similarity of the blue dye to NAD, a natural substrate for domain III of PE40 . Green and red dye-ligand chromatography steps removed nicotinamide as well as minor residual E . coli proteins from PE40 . Reversed-phase liquid chromatography peptide maps of purified PE40 were characterized by electrospray ionization mass spectrometry to determine the sequence and verify disulfide bonding.

FEMS Microbiol Lett, 1995 Aug 15, 131(1), 63 - 7
Saprophytic Pseudomonas syringae strain M1 of wheat produces cyclic lipodepsipeptides; Adetuyi FC et al.; A saprophytic fluorescent bacterium (strain M1) isolated from wheat was identified as Pseudomonas syringae and shown to produce the cyclic lipodepsipeptides, syringomycin E and syringopeptin SP25A . M1 grew in planta but did not affect germination or cause disease symptoms in wheat . The findings show that the production of these metabolites, generally regarded as plant virulence factors, does not correlate with plant pathogenicity.

Science, 1995 Aug 11, 269(5225), 843 - 6
Structure of the Arabidopsis RPM1 gene enabling dual specificity disease resistance; Grant MR et al.; Plants can recognize pathogens through the action of disease resistance (R) genes, which confer resistance to pathogens expressing unique corresponding avirulence (avr) genes . The molecular basis of this gene-for-gene specificity is unknown . The Arabidopsis thaliana RPM1 gene enables dual specificity to pathogens expressing either of two unrelated Pseudomonas syringae avr genes . Despite this function, RPM1 encodes a protein sharing molecular features with recently described single-specificity R genes . Surprisingly, RPM1 is lacking from naturally occurring, disease-susceptible Arabidopsis accessions.

Biochemistry, 1995 Aug 8, 34(31), 9930 - 5
Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase: testing the function of the active site cysteine by site-directed mutagenesis; Narasimhan C et al.; Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase is affinity labeled by 2-butynoyl-CoA; peptide sequence analysis demonstrates C237 to be the site of modification {Hruz et al . (1992) Biochemistry 31, 6842-6847} . In order to evaluate whether C237 functions in the chemistry of hydroxymethylglutaryl-CoA cleavage, cassette mutagenesis has been employed to alter wild-type DNA to encode serine or alanine at residue 237 . ESR measurements indicate that the purified mutant enzymes bind stoichiometric amounts of the spin-labeled substrate analog, R.CoA, which has been established as a competitive inhibitor . Binding affinities measured with C237S (Kd = 92 microM) and C237A (Kd = 97 microM) lyases are comparable to that observed with wild-type lyase . The rotational dynamics of R.CoA bound to mutant enzymes are also very similar to those for R.CoA bound to wild-type lyase . These observations suggest that the mutant enzymes are structurally intact . In view of this demonstrated structural integrity, it is significant that the VmaxS of C237A and C237S are approximately 4 x 10(4)- and approximately 725-fold lower, respectively, than the value measured for wild-type hydroxymethylglutaryl-CoA lyase . The C237S enzyme exhibits a Km = 53 microM for substrate; this value is only 2-fold higher than the Km of the wild-type enzyme . Additionally, we report that the residual activity in C237S hydroxymethylglutaryl-CoA lyase is unaffected by 2-butynoyl-CoA under conditions which support inactivation of wild-type enzyme . These results are consistent with an active site assignment to C237, confirming the prediction based on the affinity labeling/peptide mapping data.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1995 Aug 7, 369(2-3), 239 - 42
Selective disulphide linkage of plant thionins with other proteins; Pineiro M et al.; Thionins are shown to form disulphide linkages with other proteins . The reaction with bacterial enzymes beta-glucuronidase and neomycin phosphotransferase II could be prevented and reversed with dithiothreitol and blocked with N-ethylmaleimide . Other cysteine-rich low-molecular-weight toxic peptides from plants (LTP-3 from barley and P19 from potato) did not react as the thionins . Certain cysteine-containing proteins, such bovine serum albumin, ovalbumin and cytochrome c, reacted with thionins, while others, including carbonic anhydrase, soybean trypsin inhibitor, bovine-lung trypsin inhibitor and phosphorylase B did not . Selectivity of the reaction with a periplasmic component of the phytopathogenic bacterium Pseudomonas solanacearum was also shown.

J Biol Chem, 1995 Aug 4, 270(31), 18309 - 12
Reaction mechanism of L-2-haloacid dehalogenase of Pseudomonas sp . YL . Identification of Asp10 as the active site nucleophile by 18O incorporation experiments; Liu JQ et al.; L-2-Haloacid dehalogenase (EC 3.8.1.2) catalyzes the hydrolytic dehalogenation of L-2-haloacids to produce the corresponding D-2-hydroxy acids . We have analyzed the reaction mechanism of the enzyme from Pseudomonas sp . YL and found that Asp10 is the active site nucleophile . When the multiple turnover enzyme reaction was carried out in H2(18)O with L-2-chloropropionate as a substrate, lactate produced was labeled with 18O . However, when the single turnover enzyme reaction was carried out by use of a large excess of the enzyme, the product was not labeled . This suggests that an oxygen atom of the solvent water is first incorporated into the enzyme and then transferred to the product . After the multiple turnover reaction in H2(18)O, the enzyme was digested with lysyl endopeptidase, and the molecular masses of the peptide fragments formed were measured by an ionspray mass spectrometer . Two 18O atoms were shown to be incorporated into a hexapeptide, Gly6-Lys11 . Tandem mass spectrometric analysis of this peptide revealed that Asp10 was labeled with two 18O atoms . Our previous site-directed mutagenesis experiment showed that the replacement of Asp10 led to a significant loss in the enzyme activity . These results indicate that Asp10 acts as a nucleophile on the alpha-carbon of the substrate leading to the formation of an ester intermediate, which is hydrolyzed by nucleophilic attack of a water molecule on the carbonyl carbon atom.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1995 Aug, 17(4), 248 - 52
{Construction and expression of the gene CD4V1V2-PE40, coding for a molecular targeted protein against AIDS}; Zhao X et al.; We here replaced the cell-binding domain in Pseudomonas exotoxin A (PE) with HIV-binding portion of CD4V1V2 molecule and constructed a chimeric gene CD4V1V2-PE40, which can be expressed in E . coli . The hybrid protein displays targeting toxicity toward cells infected by HIV and thus represents a promising novel therapeutic agent for the treatment of AIDS.

Plant J, 1995 Aug, 8(2), 235 - 45
Hydrogen peroxide does not function downstream of salicylic acid in the induction of PR protein expression; Bi YM et al.; The roles of salicylic acid (SA) and H2O2 in the induction of PR proteins in tobacco have been examined . Studies were conducted on wild-type tobacco and plants engineered to express a bacterial salicylate hydroxylase capable of metabolizing SA to catechol (SH-L plants) . Wild-type and PR-1a-GUS-transformed plants express PR-1a following challenge with Pseudomonas syringae pathovar syringae, SA or 2,6-dichloro-isonicotinic acid (INA) . In contrast, SH-L plants failed to respond to SA but did express PR-1a following INA treatment . H2O2 and the irreversible catalase inhibitor 3-amino-1,2,4-triazole (3-AT) were found to be weak inducers of PR-1a expression (relative to SA) in wild-type tobacco but were unable to induce PR-1a in SH-L plants, suggesting that the action of these compounds depends upon the accumulation of SA . A model has been proposed suggesting that SA binds to and inhibits a catalase inducing an increase in H2O2 leading to PR protein expression . Catalase activity has been measured in tobacco and no significant changes in activity following infection with P . syringae pv . syringae were detected . Furthermore, inhibition of catalase activity in vitro in plant extracts requires pre-incubation and only occurs at SA concentrations above 250 microM . Leaf disks preincubated with 1 mM SA do accumulate SA to these levels and PR-1a is efficiently induced but there is no apparent inhibition of catalase activity . It is also shown that a SA-responsive gene, PR-1a, and a H2O2-sensitive gene, AoPR-1, are both relatively insensitive to 3-AT suggesting that induction of these genes is unlikely to be due entirely to inhibition of an endogenous catalase.

Mol Pharmacol, 1995 Aug, 48(2), 206 - 11
Regulation of beta 2-adrenergic receptor mRNA and gene transcription in rat C6 glioma cells: effects of agonist, forskolin, and protein synthesis inhibition; Hosoda K et al.; Incubation of rat C6 glioma cells with beta-adrenergic receptor (beta AR) agonist or with agents that increase cAMP levels results in down-regulation of the beta 2AR, as measured by the loss of radioligand binding sites . In the present study, the role of beta 2AR mRNA expression and stability in the down-regulation of beta 2AR sites in C6 cells was examined . Isoproterenol or forskolin treatment decreased beta 2AR mRNA levels in a time-dependent manner, with maximal loss of approximately 50% being observed after 2 hr . Pretreatment of the cells with a potent protein synthesis inhibitor, Pseudomonas exotoxin A, completely blocked isoproterenol- and forskolin-mediated down-regulation of beta 2AR mRNA . Exposure to agonist did not significantly influence the half-life of beta 2AR mRNA, which was approximately 60 min . In contrast, isoproterenol treatment for 2 hr significantly decreased the rate of beta 2AR gene transcription, as determined by nuclear run-on analysis . Based on these results, we propose that agonist regulation of beta 2AR mRNA in C6 cells is mediated by activation of the cAMP system and occurs at the level of beta 2AR gene transcription, not mRNA stability . In addition, the observed requirement for protein synthesis indicates that down-regulation of beta 2AR mRNA may be mediated by expression of a repressor of beta 2AR gene transcription.

J Bacteriol, 1995 Aug, 177(16), 4658 - 68
Identification and characterization of a locus which regulates multiple functions in Pseudomonas tolaasii, the cause of brown blotch disease of Agaricus bisporus; Grewal SI et al.; Pseudomonas tolaasii, the causal agent of brown blotch disease of Agaricus bisporus, spontaneously gives rise to morphologically distinct stable sectors, referred to as the phenotypic variant form, at the margins of the wild-type colonies . The phenotypic variant form is nonpathogenic and differs from the wild type in a range of biochemical and physiological characteristics . A genomic cosmid clone (pSISG29) from a wild-type P . tolaasii library was shown to be capable of restoring a range of characteristics of the phenotypic variant to those of the wild-type form, when present in trans . Subcloning and saturation mutagenesis analysis with Tn5lacZ localized a 3.0-kb region from pSISG29, designated the pheN locus, required for complementation of the phenotypic variant to the wild-type form . Marker exchange of the Tn5lacZ-mutagenized copy of the pheN locus into the wild-type strain demonstrated that a functional copy of the pheN gene is required to maintain the wild-type pathogenic phenotype and that loss of the pheN gene or its function results in conversion of the wild-type form to the phenotypic variant form . The pheN locus contained a 2,727-bp open reading frame encoding an 83-kDa protein . The predicted amino acid sequence of the PheN protein showed homology to the sensor and regulator domains of the conserved family of two component bacterial sensor regulator proteins . Southern hybridization analysis of pheN genes from the wild type and the phenotypic variant form revealed that DNA rearrangement occurs within the pheN locus during phenotypic variation . Analysis of pheN expression with a pheN::lacZ fusion demonstrated that expression is regulated by environmental factors . These results are related to a model for control for phenotypic variation in P . tolaasii.

Plant Mol Biol, 1995 Aug, 28(5), 871 - 84
The Arabidopsis thaliana 4-coumarate:CoA ligase (4CL) gene: stress and developmentally regulated expression and nucleotide sequence of its cDNA; Lee D et al.; An Arabidopsis cDNA clone encoding 4-coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism, was identified and sequenced . The predicted amino acid sequence is similar to those of other cloned 4CL genes . Southern blot analysis indicated that 4CL is single-copy gene in Arabidopsis . Northern blots showed that 4CL expression was activated early during seedling development . The onset of 4CL expression was correlated with the onset of lignin deposition in cotyledons and roots 2-3 days after germination . The timing of the expression of a parsley 4CL1-GUS fusion in transgenic Arabidopsis seedlings was examined in parallel and was very similar to that of endogenous 4CL . In mature plants, highest 4CL expression was observed in bolting stems, where relatively large amounts of lignin accumulate . Both 4CL and 4CL1-GUS mRNA accumulation was strongly and transiently activated by wounding of mature Arabidopsis leaves . 4CL expression was specifically activated within 6 h after infiltration of Arabidopsis ecotype Columbia leaves with a Pseudomonas syringae pv . maculicola strain harboring the bacterial avirulence gene avrB, which causes in incompatible interaction . The timing of 4CL activation was identical to the previously observed activation of PAL gene expression in this interaction . No activation of 4CL expression was observed in a compatible interaction caused by a Pseudomonas syringae pv . maculicola strain without avrB.

Lett Appl Microbiol, 1995 Aug, 21(2), 87 - 92
Construction of species-specific primers for Pseudomonas andropogonis based on 16S rDNA sequences; Bagsic RD et al.; Approximately 94% of the total 16S rDNA of Pseudomonas andropogonis strain ACH 01053A was sequenced and compared with that of strain ATCC 23061 obtained from the GenBank database . The two sequences were highly homologous with 1.3% difference . Alignment of sequences with those from closely related bacteria revealed two possible regions for design of a specific primer suitable for detection of Ps . andropogonis . Using primers designed to these variable regions in a PCR test, an amplification product of approximately 410 bp was specifically produced by 40 strains of Ps . andropogonis . No other bacterial species showed an amplification product under optimized PCR conditions . As few as 1000 cells per reaction were detected.

Infect Immun, 1995 Aug, 63(8), 2912 - 8
Level of receptor-associated protein moderates cellular susceptibility to pseudomonas exotoxin A; Mucci D et al.; Pseudomonas exotoxin A (PE) enters mammalian cells via a receptor-mediated endocytic pathway . The initial step in this pathway is binding to the multiligand receptor termed the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein (LRP) . Binding of toxin, and of the many other ligands that bind to LRP, is blocked by the addition of a 39-kDa receptor-associated protein (RAP) . Here we show that approximately 40% of the cell-associated LRP is on the surface of toxin-sensitive mouse LM fibroblasts and thus accessible for toxin internalization . The remainder is located intracellularly, primarily in the Golgi region . Mammalian cells exhibit a wide range of sensitivity to PE . To investigate possible reasons for this, we examined the expression levels of both LRP and RAP . Results from a variety of cell lines indicated that there was a positive correlation between LRP expression and toxin sensitivity . In the absence of LRP, cells were as much as 200-fold more resistant to PE compared with sensitive cells . A second group of resistant cells expressed LRP but had a high level of RAP . Thus, a toxin-resistant phenotype would be expected when cells expressed either low levels of LRP or high levels of LRP in the presence of high levels of RAP . We hypothesize that RAP has a pivotal role in moderating cellular susceptibility to PE.

Cancer Res, 1995 Aug 1, 55(15), 3357 - 63
Increased antitumor activity of a circularly permuted interleukin 4-toxin in mice with interleukin 4 receptor-bearing human carcinoma; Kreitman RJ et al.; We reported previously that circularly permuted interleukin-4 (IL4), composed of amino acids 38-129 of IL4 connected by a linker peptide GGNGG to amino acids 1-37, is preferable to native IL4 for fusing to the amino terminus of truncated Pseudomonas exotoxin (PE) to make a recombinant toxin, because the new ligand-toxin junction results in improved IL4 receptor (IL4R)-binding (R . J . Kreitman et al., Proc . Natl . Acad . Sci . USA, 91: 6889-6893, 1994) . We now report that the improved binding of circularly permuted IL4-toxin is associated with improved antitumor activity in tumor-bearing mice . For in vivo testing, we made an improved circularly permuted IL4-toxin, termed IL4(38-37)-PE38KDEL . It contains an N38D mutation at the amino terminus, allowing improved expression and large-scale production in Escherichia coli . It also contains the truncated toxin PE38KDEL, which is composed of amino acids 253-364 and 381-608 of PE, followed by KDEL . To evaluate antitumor activity, nude mice carrying s.c . tumors composed of IL4R-bearing human A431 epidermoid carcinoma cells were injected with recombinant toxins i.v . every other day for three doses . IL4(38-37)-PE38KDEL induced complete remissions in 80% of mice receiving 50 micrograms/kg x 3 and 100% of mice receiving 100 micrograms/kg x 3, while only 70% of mice receiving 200 micrograms/kg x 3 of the native IL4-toxin IL4-PE38KDEL obtained complete remission . Disease-free survival after obtaining complete remissions was higher in mice treated with IL4(38-37)-PE38KDEL 50 micrograms/Kg QOD x 3 than with IL4-PE38KDEL 200 micrograms/Kg QOD x 3 (P < 0.03) . IL4(38-37)-PE38KDEL and IL4-PE38KDEL exhibited similar toxicity and pharmacokinetics in the mice, indicating that the improved antitumor activity of the circularly permuted IL4-toxin was due to its improved binding to the IL4R on the target cells.

Exp Cell Res, 1995 Aug, 219(2), 671 - 8
Ilimaquinone inhibits the cytotoxicities of ricin, diphtheria toxin, and other protein toxins in Vero cells; Nambiar MP et al.; Ilimaquinone (IQ), a metabolite from sea sponges, has been shown to cause the breakdown of Golgi membranes into small vesicular structure and to inhibit protein transport without eliciting the retrograde transport of the Golgi enzymes to the endoplasmic reticulum {P . A . Takizawa, J . K . Yucel, B . Viet, D . J . Faulkner, T . Deerinck, G . Soto, M . Ellismann, and V . Malhotra, Cell (1993) 73, 1079-1090} . We have found that incubation of Vero cells with IQ inhibited the cytotoxicity of ricin in a dose-dependent manner . The inhibition was reversed upon the removal of IQ . Neither binding and internalization of 125I-ricin nor the translocation of ricin to the cytosol was affected by IQ . However, IQ significantly inhibited the recycling and degradation of internalized 125I-ricin . Preincubation with IQ also prevented the enhancement of ricin cytotoxicity by NH4Cl or nigericin . The inhibition of ricin cytotoxicity by IQ was observed in the presence of cycloheximide, indicating that de novo protein synthesis is not required for IQ-mediated protection of Vero cells from ricin cytotoxicity . In contrast to perinuclear distribution of TRITC-labeled ricin in Vero cells, TRITC-ricin appeared in numerous small vesicles dispersed throughout the cytoplasm in IQ-treated Vero cells . Double labeling with C6-NBD-ceramide and TRITC-labeled ricin showed that these ricin-containing vesicles were distinct from the IQ-induced breakdown product of the Golgi membranes . Like brefeldin A (BFA), IQ inhibited the cytotoxicities of abrin, modeccin, Pseudomonas toxin, and Shiga-like toxin in Vero cells . Unlike BFA, IQ also inhibited the cytotoxicity of diphtheria toxin (DT) . Inhibition of DT cytotoxicity was the consequence of a decreased specific binding of the toxin in the IQ-treated cells.

Australas Radiol, 1995 Aug, 39(3), 260 - 4
Imaging patterns in melioidosis; Tan AP et al.; Melioidosis is an infectious disease caused by Pseudomonas pseudomallei . It is seldom diagnosed promptly and, if untreated, can lead to an 80-100% mortality rate . Twenty-eight patients with melioidosis were identified over a 6 year period, and their imaging patterns were analysed . Respiratory infections were the commonest form of presentation, frequently shown as diffuse airspace consolidation, and accounted for the highest mortality . Visceral and musculoskeletal infections were associated with chronicity and a high relapse rate . Multifocal splenic abscesses were a common occurrence . Septic arthritis of the knee was frequently seen . The majority of patients had diabetes mellitus and chronic ill-health . An increased awareness of the disease can contribute to its early detection and appropriate treatment.

Int J Cancer, 1995 Jul 28, 62(3), 351 - 5
Administration of disulfide-stabilized Fv-immunotoxins B1(dsFv)-PE38 and B3(dsFv)-PE38 by continuous infusion increases their efficacy in curing large tumor xenografts in nude mice; Benhar I et al.; B1 (dsFv)-PE38 and B3(dsFv)-PE38 are recombinant immunotoxins in which the Fv fragments of MAbs B1 and B3, respectively, are stabilized by an engineered interchain disulfide bond and are fused at their C-termini to a modified Pseudomonas exotoxin from which the cell-binding domain has been deleted (PE38) . Both immunotoxins have been shown to be specifically cytotoxic toward human cancer cell lines which express Le gamma-related carbohydrates on their surface, and when given i.v., eradicated 30- to 50-mm3 s.c . A431 tumors growing in nude mice . A major advantage of dsFv-immunotoxins is their stability at 37 degrees C compared with the relatively unstable single-chain Fvs . This allows them to be given continuously by osmotic pumps placed in the peritoneal cavity . In an attempt to increase the therapeutic index of the immunotoxins, we have now delivered them continuously for 6 days through mini-osmotic pumps placed in the peritoneal cavity of tumor-bearing nude mice . Using this mode of administration, we were able to maintain a constant level of immunotoxin in the serum which was non-toxic to the mice, but caused complete regressions of large 150- to 200-mm3 tumors which lasted for over a month at 1/11 of the LD50 with B1(dsFv)-PE38 and 1/6 of the LD50 with B3(dsFv)-PE38 . Complete regression of tumors of similar size could also be achieved by i.v . bolus injections of these immunotoxins at 1/7 of the LD50 with B1(dsFv)-PE38) and 1/3 of the LD50 with B3(dsFv)-PE38 . These results suggest that in patients it may be advantageous to administer dsFv-immunotoxins by continuous infusion, since a larger therapeutic index is achieved.

Cancer Res, 1995 Jul 15, 55(14), 3099 - 104
Activity of a single-chain immunotoxin that selectively kills lymphoma and other B-lineage cells expressing the CD40 antigen; Francisco JA et al.; We have constructed anti-CD40 immunotoxins consisting of the single chain Fv (sFv) region of the anti-human CD40 mAb G28-5 fused to a truncated form of Pseudomonas exotoxin, PE40 . CD40 is an integral membrane glycoprotein found on the surface of B-lineage cells, including lymphomas and leukemias, as well as certain carcinomas . Two forms of the immunotoxin were constructed, one with the light chain variable (VL) region of the sFv preceding the heavy chain variable region (VH) {G28-5 sFv(VL-VH)-PE40} and the second with the sFv in the opposite orientation {G28-5 sFv(VH-VL)-PE40} . Although both forms of G28-5 sFv-PE40 specifically bound to CD40 in ELISAs, the binding of G28-5 sFv(VL-VH)-PE40 was > 10-fold higher . A number of malignant B- and T-cell lines were screened for CD40 expression and susceptibility to G28-5 sFv(VL-VH)-PE40 . All of the B-lineage cells tested were CD40 positive and sensitive to the anti-CD40 immunotoxin, with EC50s ranging from 2.5-70 ng/ml, whereas none of the T cell leukemias or lymphomas were antigen positive or were affected by the immunotoxins . Consistent with the antigen-binding results, the VL-VH immunotoxin was > 10-fold more cytotoxic than the VH-VL immunotoxin . The anti-CD40 single-chain immunotoxin fusion protein G28-5 sFv(VL-VH)-PE40 represents a potent and specific cytotoxic agent for the elimination of normal and transformed B-lineage cells expressing CD40.

J Biol Chem, 1995 Jul 14, 270(28), 16775 - 80
A novel chimeric protein composed of interleukin 13 and Pseudomonas exotoxin is highly cytotoxic to human carcinoma cells expressing receptors for interleukin 13 and interleukin 4; Debinski W et al.; Chimeric proteins provide a unique opportunity to target therapeutic bacterial toxins to a subset of specific cells . We have generated a new recombinant chimeric toxin composed of human interleukin 13 (hIL13) and a Pseudomonas exotoxin A (PE) mutant, PE38QQR . The hIL13-PE38QQR chimera is highly cytotoxic to cell lines derived from several human epithelial carcinomas such as adenocarcinoma of stomach, colon, and skin . The cytotoxic action of hIL13-PE38QQR, which can only occur upon internalization of ligand-receptor complex, is blocked by an excess of hIL13 but not of hIL2 . This action is not solely hIL13-specific because an excess of hIL4 also blocks the cytotoxicity of hIL13-toxin . Conversely, hIL13 blocks the cytotoxicity of a hIL4-PE38QQR chimera . Binding studies showed that hIL13 displaces competitively 125I-labeled hIL4-PE38QQR on carcinoma cells . These results indicate that IL4 and IL13 compete for a common binding site on the studied human cell lines . Despite this competition, hIL4 but not hIL13 decreased protein synthesis in malignant cells susceptible to the cytotoxicity of both hIL13- and hIL4-PE38QQR . Our results suggest that a spectrum of human carcinomas express binding sites for IL13 . Furthermore, hIL13 and hIL4 compete reciprocally for a form of the receptor that is internalized upon binding a ligand . Thus, cancer cells represent an interesting model for studying receptors for these two growth factors . Finally, hIL13-PE38QQR may be a useful agent in the treatment of several malignancies.

Mol Plant Microbe Interact, 1995 Jul-Aug, 8(4), 637 - 40
A disease resistance gene in Arabidopsis with specificity for the avrPph3 gene of Pseudomonas syringae pv . phaseolicola; Simonich MT et al.; The avirulence gene avrPph3 from Pseudomonas syringae pv . phaseolicola was tested for its ability to convert virulent P . syringae pv . tomato strain DC3000 to avirulence on Arabidopsis . In F2 plants from a cross between resistant and susceptible ecotypes, the ratio of resistant to susceptible plants was approximately 3:1, indicating that resistance to DC3000(avrPph3) is determined by a single dominant locus, which we have designated RPS5 . RPS5 was mapped to chromosome 1, between restriction fragment length polymorphism markers m241 and g3786.

Mol Plant Microbe Interact, 1995 Jul-Aug, 8(4), 610 - 20
Role of biosurfactant and ion channel-forming activities of syringomycin in transmembrane ion flux: a model for the mechanism of action in the plant-pathogen interaction; Hutchison ML et al.; Syringomycin is a necrosis-inducing lipopeptide toxin synthesized and secreted by the phytopathogen, Pseudomonas syringae pv . syringae . Although small quantities of syringomycin are known to activate a cascade of physiological events in plasma membranes, the mechanism of action of the phytotoxin has never been fully characterized . The objective of this study was to test the hypothesis that the primary mode of action of syringomycin is to form transmembrane pores that are permeable to cations . Accordingly, direct measurement of ion fluxes were performed using artificial bilayers . The hemolytic properties and surface activity of HPLC-purified syringomycin were quantified by use of an erythrocyte lysis assay and by the drop weight method . Assays were performed using syringomycin form SRE alone or a mixture containing all forms of the phytotoxin . At a threshold concentration of 500 ng/ml, syringomycin induced hemolysis by forming ion channels in membranes . Osmotic protection studies indicated a channel radius of between 0.6 and 1 nm . The ion channel-forming activity was insensitive and permeable to both monovalent and divalent cations, suggesting that syringomycin causes lysis of erythrocytes by colloid osmotic lysis . In addition, syringomycin, like other lipopeptide antibiotics, is a potent biosurfactant capable of lowering the interfacial tension of water to 31 mN/m . The critical micellar concentration of syringomycin was calculated to be 1.25 mg/ml and the gamma CMC was 33 mN/m . A model is presented depicting the mechanism of action of syringomycin in the plant-pathogen interaction . The model integrates known effects of the toxin on ion flux in plasma membranes with formation of ion channels and the consequential cascade of effects associated with cellular signalling.

J Antimicrob Chemother, 1995 Jul, 36 Suppl A, 135 - 43
Clinical evaluation of meropenem versus ceftazidime for the treatment of Pseudomonas spp . infections in cystic fibrosis patients; Byrne S et al.; Cystic fibrosis patients (children and young adults) with Pseudomonas spp . chest infections were treated with meropenem or ceftazidime . This study was the first to investigate the use of meropenem in cystic fibrosis . Meropenem was well tolerated with only transient elevations of serum transaminases . No patient experienced nausea and vomiting, even when meropenem was administered as a bolus injection . This allowed home therapy to be used . Meropenem appeared to be at least as active as ceftazidime even at the low doses used . Patients showed a greater improvement in respiratory function on meropenem than ceftazidime . Only one patient (out of 60 courses) failed to respond to meropenem (98% success rate) compared with two failures out of 21 episodes with ceftazidime (90% success rate) . There was little emergence of resistance to meropenem even though some patients were treated up to eight times over a 2 year period.

Biosci Biotechnol Biochem, 1995 Jul, 59(7), 1374 - 6
Structural analysis of new syringopeptins by tandem mass spectrometry; Isogai A et al.; New syringopeptins SP(SC)-1 and -2 were isolated from culture filtrates of phytopathogenic bacterium strain SC1 of Pseudomonas syringae pv . syringae . These syringopeptins were composed of a beta-hydroxy fatty acid, a long sequence of aliphatic amino acids . and a lactone moiety of eight amino acids . The amino acid sequences were deduced from a comparison of their tandem mass sepctra with those of known syringopeptins SP-22a and SP-25a . SP(SC)-1 and SP(SC)-2 resembled SP-22a, but differed from the latter by 3 amino acids.

Biosci Biotechnol Biochem, 1995 Jul, 59(7), 1331 - 2
Cloning of the creatinine amidohydrolase gene from Pseudomonas sp . PS-7; Yamamoto K et al.; The gene encoding creatinine amidohydrolase (EC 3.5.2.10) was isolated from Pseudomonas sp . PS-7 . The primary structure of creatinine amidohydrolase deduced from the nucleotide sequence showed that the protein is composed of 259 amino acids and has a molecular weight of 28,437 . The enzymatic property of creatinine amidohydrolase produced by recombinant E . coli was identical with those by Pseudomonas sp . PS-7.

Appl Microbiol Biotechnol, 1995 Jul, 43(3), 498 - 507
A target-specific chimeric toxin composed of epidermal growth factor and Pseudomonas exotoxin A with a deletion in its toxin-binding domain; Liao CW et al.; We have fused the epidermal growth factor (EGF) to the amino terminus of Pseudomonas exotoxin A (PE) to create a cytotoxic agent, designated EGF-PE, which preferentially kills EGF-receptor-bearing cells . In this study, we analyzed the effect of the Ia domain, the binding domain of PE on the cytotoxicity of EGF-PE towards EGF-receptor-bearing cells and tried to develop a more potent EGF-receptor-targeting toxin . EGF-PE molecules with sequential deletions at the amino terminus of PE were constructed and expressed in E . coli strain BL21(DE3) . The cytotoxicity of these chimeric toxins was then examined . Our results show that the amino-terminal and carboxy-terminal regions of the Ia domain of PE are important for the cytotoxicity of a PE-based targeting toxin . To design a more potent PE-based EGF-receptor-targeting toxin, a chimeric toxin, named EGF-PE(delta 34-220), which had most of the Ia domain deleted but retained amino acid residues 1-33 and 221-252 of this domain, was constructed . EGF-PE(delta 34-220) has EGF-receptor-binding activity but does not show PE-receptor-binding activity and is mildly cytotoxic to EGF-receptor-deficient NR6 cells . As expected, EGF-PE(delta 34-220) is a more potent cytotoxic agent towards EGF-receptor-bearing cells than EGF-PE(delta 1-252), where the entire Ia domain of PE was deleted . In addition, EGF-PE(delta 34-220) was shown to be extremely cytotoxic to EGF-receptor-bearing cancer cells, such as A431, CE81T/VGH, and KB-3-1 cells . We also found that EGF-PE(delta 34-220) was highly expressed in BL21(DE3) and could be easily purified by urea extraction . Thus, EGF-PE(delta 34-220) can be a useful cytotoxic agent towards EGF-receptor-bearing cells.

Eur J Biochem, 1995 Jul 1, 231(1), 133 - 41
Electron transfer and spectral alpha-band properties of the di-heme protein cytochrome c4 from Pseudomonas stutzeri; Conrad LS et al.; Cytochrome c4 is a 190-residue protein active in the aerobic and anaerobic respiration of several bacteria . We have isolated Pseudomonas stutzeri (ATCC no . 11607) cytochrome c4 by an optimized growth procedure following factorial design . The ultraviolet/visible spectra of reduced cytochrome c4 have a composite alpha/beta band which can be resolved into six components . One of these seems to be specific for the high-potential heme group . The kinetics for full oxidation and reduction with the two inorganic redox couples, {Co(terpy)2}2+/3+ and {Co(bipy)3}2+/3+, is formally compatible with either bi- or tri-exponential kinetics . The former would be in line with weak interaction between the heme groups, the latter with notable interaction effects . Arguments in favour of the latter and a cooperative two-electron transfer pattern are given . All phases are approximately proportional to the Co-complex concentration, implying that intramolecular electron transfer in this time range is unlikely . The rate constants are in the range (0.7-80) x 10(4) M-1 s-1 at pH = 7.6 (Tris) and 0.1 M NaCl and very little dependent on the ionic strength in the range 0.1-0.3 M . The reduction potentials could be calculated from the forward and reverse rate constant ratios . The values are 241 +/- 5 and 328 +/- 2 mV (Nernst hydrogen electrode) if bi-exponential kinetics is used and interaction between the heme groups disregarded . The intrinsic microscopic reduction potential values are closer when the tri-exponential, cooperative model is used as this model transfers 30-40 mV to electrostatically dominated interaction potentials . The overall electron transfer pattern can be related to the recently determined crystal structure of the P . stutzeri cytochrome c4.

Appl Environ Microbiol, 1995 Jul, 61(7), 2654 - 8
Conversion of chlorobiphenyls into phenylhexadienoates and benzoates by the enzymes of the upper pathway for polychlorobiphenyl degradation encoded by the bph locus of Pseudomonas sp . strain LB400; Seeger M et al.; Metabolism of 21 chlorobiphenyls by the enzymes of the upper biphenyl catabolic pathway encoded by the bph locus of Pseudomonas sp . strain LB400 was investigated by using recombinant strains harboring gene cassettes containing bphABC or bphABCD . The enzymes of the upper pathway were generally able to metabolize mono- and dichlorinated biphenyls but only partially transform most trichlorinated congeners investigated: 14 of 15 mono- and dichlorinated and 2 of 6 trichlorinated congeners were converted into benzoates . All mono- and at least 8 of 12 dichlorinated congeners were attacked by the bphA-encoded biphenyl dioxygenase virtually exclusively at ortho and meta carbons . This enzyme exhibited a high degree of selectivity for the aromatic ring to be attacked, with the order of ring preference being non- > ortho- > meta- > para-substituted for mono- and dichlorinated congeners . The influence of the chlorine substitution pattern of the metabolized ring on benzoate formation resembled its influence on the reactivity of initial dioxygenation, suggesting that the rate of benzoate formation may frequently be determined by the rate of initial attack . The absorption spectra of phenylhexadienoates formed correlated with the presence or absence of a chlorine substituent at an ortho position.

J Bacteriol, 1995 Jul, 177(13), 3837 - 42
Purification and characterization of nitrobenzene nitroreductase from Pseudomonas pseudoalcaligenes JS45; Somerville CC et al.; Pseudomonas pseudoalcaligenes JS45 grows on nitrobenzene as a sole source of carbon, nitrogen, and energy . The catabolic pathway involves reduction to hydroxylaminobenzene followed by rearrangement to o-amino-phenol and ring fission (S . F . Nishino and J . C . Spain, Appl . Environ . Microbiol . 59:2520, 1993) . A nitrobenzene-inducible, oxygen-insensitive nitroreductase was purified from extracts of JS45 by ammonium sulfate precipitation followed by anion-exchange and gel filtration chromatography . A single 33-kDa polypeptide was detected by denaturing gel electrophoresis . The size of the native protein was estimated to be 30 kDa by gel filtration . The enzyme is a flavoprotein with a tightly bound flavin mononucleotide cofactor in a ratio of 2 mol of flavin per mol of protein . The Km for nitrobenzene is 5 microM at an initial NADPH concentration of 0.5 mM . The Km for NADPH at an initial nitrobenzene concentration of 0.1 mM is 183 microM . Nitrosobenzene was not detected as an intermediate of nitrobenzene reduction, but nitrosobenzene is a substrate for the enzyme, and the specific activity for nitrosobenzene is higher than that for nitrobenzene . These results suggest that nitrosobenzene is formed but is immediately reduced to hydroxylaminobenzene . Hydroxylaminobenzene was the only product detected after incubation of the purified enzyme with nitrobenzene and NADPH . Hydroxylaminobenzene does not serve as a substrate for further reduction by this enzyme . The products and intermediates are consistent with two two-electron reductions of the parent compound . Furthermore, the low Km and the inducible control of enzyme synthesis suggest that nitrobenzene is the physiological substrate for this enzyme.

J Thorac Cardiovasc Surg, 1995 Jul, 110(1), 22 - 6
Pleural complications in lung transplant recipients; Herridge MS et al.; Pleural complications occurred in 30 (22%) of 138 patients after 53 single and 91 double lung transplants between September 1986 and February 1993 . These were defined for the purpose of this study as pneumothorax persisting beyond the first 14 postoperative days, recurrent pneumothorax, or any other pleural process that necessitated diagnostic or therapeutic intervention . Overall, a higher pleural complication rate was seen in double lung transplantation (25 of 30) than in single lung transplantation (5 of 30) with no differences noted in the frequency among preoperative diagnostic groups (p > 0.05) . Pneumothorax was the most frequent complication, affecting 14 of 30 patients, with 6 of 14 cases occurring after transbronchial biopsy . All pneumothoraces in single (n = 4) and double lung transplantation (n = 10) resolved spontaneously or with chest tube thoracostomy . One patient required placement of a Clagett window after open lung biopsy and another required thoracotomy and pleural abrasion after transbronchial biopsy . Parapneumonic effusion was observed in 4 of 30 double lung transplantations with spontaneous resolution in all cases . Empyema affected 7 of 30 patients and occurred exclusively in the double lung transplant group . Sepsis developed in three of the patients with this complication and they subsequently died . The risk of empyema was independent of preoperative diagnosis (p > 0.05) . Of interest, all patients with cystic fibrosis (n = 3) with complicating empyema had Pseudomonas cepacia in the pleural fluid . Other miscellaneous complications included subpleural hematoma, chylothorax, and hemothorax . The latter two necessitated thoracic duct and bronchial artery ligation, respectively . In summary, a significant proportion of lung transplant recipients will have pleural space complications . The vast majority of these will resolve spontaneously or with conservative procedures . These complications were not related to preoperative diagnosis nor associated with a significant prolongation of hospital stay (p > 0.05) . Empyema is the only pleural space complication associated with increased patient mortality and, as such, is an important clinical marker for those at risk for sepsis and death.

Antimicrob Agents Chemother, 1995 Jul, 39(7), 1569 - 73
Product of fosC, a gene from Pseudomonas syringae, mediates fosfomycin resistance by using ATP as cosubstrate; Garcia P et al.; Pseudomonas syringe PB-5123, a producer of fosfomycin, is resistant to high concentrations of the antibiotic . Two possible mechanisms of resistance have been detected: (i) impermeability to exogenous fosfomycin, even in the presence of sugar phosphate uptake inducers, and (ii) antibiotic phosphorylation . The gene responsible for this last activity, fosC, encodes a ca . 19,000-Da protein and is immediately followed by a second open reading frame, which shows sequence similarities to glutathione S-transferases . FosC uses ATP as a cosubstrate in an inactivation reaction that can be reversed with alkaline phosphatase . Other nucleotide triphosphates cannot be substituted for ATP in this reaction . No relationship between fosC and the previously described genes of fosfomycin resistance was found.

Proteins, 1995 Jul, 22(3), 284 - 6
Crystallization and preliminary crystallographic analysis of a 2,3-dihydroxybiphenyl dioxygenase from Pseudomonas sp . strain KKS102 having polychlorinated biphenyl (PCB)-degrading activity; Sugiyama K et al.; Crystals have been obtained for a 2,3-dihydroxybiphenyl dioxygenase (conventionally called BphC) from a polychlorinated biphenyl (PCB)-degrader, Pseudomonas sp . strain KKS102 . The crystals were grown using both ammonium sulfate and MPD as the precipitating agents . The crystals belonged to a tetragonal space group (I422) and diffracted to 2.5 A.

J Biol Chem, 1995 Jun 30, 270(26), 15747 - 54
Enhancement of the binding of triglyceride-rich lipoproteins to the very low density lipoprotein receptor by apolipoprotein E and lipoprotein lipase; Takahashi S et al.; The low-density lipoprotein (LDL) receptor plays a crucial role in cholesterol metabolism . A related protein, designated the very low density lipoprotein (VLDL) receptor, that specifically binds apolipoprotein (apo) E has recently been characterized and shown to be expressed in heart, muscle and adipose tissue and the human monocyte-macrophage cell line THP-1 . The VLDL receptor binds and internalizes VLDL and intermediate density lipoprotein from Watanabe heritable hyperlipidemic (WHHL) rabbits as well as beta-migrating VLDL from cholesterol-fed rabbits but not LDL from WHHL rabbits . Chinese hamster ovary (CHO) cells transfected with the rabbit VLDL receptor cDNA have now been shown to bind or internalize VLDL (d < 1.006 g/ml) isolated from fasted normolipidemic human subjects with lower affinity than WHHL-VLDL or rabbit beta-VLDL . However, binding and internalization were markedly enhanced when fasted human VLDL was preincubated with either recombinant human apoE (3/3) or lipoprotein lipase (LPL) in CHO cells overexpressing the rabbit or human VLDL receptor . CHO cells transfected with both the rabbit VLDL receptor cDNA and the human LPL cDNA effectively bound, internalized, and degraded fasted human VLDL without pretreatment . Treatment of heparinase reduced the effect of LPL-mediated binding at 4 degrees C, but the inhibitory effect was lower at 37 degrees C . Pseudomonas LPL also enhanced the binding of human fasted VLDL to the VLDL receptor at 37 degrees C in CHO cells overexpressing the human VLDL receptor . Taken together, LPL causes the enhancement of triglyceride-rich lipoproteins binding to the VLDL receptor via both the formation of bridge between lipoproteins and heparan sulfate proteoglycans and its lipolytic effect . Ligand blot analysis showed that the apparent molecular mass of the VLDL receptor is 118 kDa, which is smaller than that of the LDL receptor . These results indicate that the VLDL receptor recognizes both triglyceride-rich lipoproteins that are also relatively rich in apoE, as well as the remnants of triglyceride-rich lipoproteins after catabolism and the interaction with heparan sulfate proteoglycans by LPL . The VLDL receptor may thus function as a receptor for remnants of triglyceride-rich lipoproteins in extrahepatic tissues.

Biochemistry, 1995 Jun 27, 34(25), 7973 - 8
Three-dimensional structure of the binuclear metal center of phosphotriesterase; Benning MM et al.; Phosphotriesterase, as isolated from Pseudomonas diminuta, is capable of detoxifying widely used pesticides such as paraoxon and parathion and various mammalian acetylcholinesterase inhibitors . The enzyme requires a binuclear metal center for activity . Recently, the three-dimensional structure of the apoenzyme was solved (Benning et al., 1994) and shown to consist of an alpha/beta-barrel . Here we describe the three-dimensional structure of the holoenzyme, reconstituted with cadmium, as determined by X-ray crystallographic analysis to 2.0-A resolution . Crystals employed in the investigation belonged to the space group C2 with unit cell dimensions of a = 129.5 A, b = 91.4 A, c = 69.4 A, beta = 91.9 degrees, and two subunits in the asymmetric unit . There are significant differences in the three-dimensional architecture of the apo and holo forms of the enzyme such that their alpha-carbon positions superimpose with a root-mean-square deviation of 3.4 A . The binuclear metal center is located at the C-terminus of the beta-barrel with the cadmiums separated by 3.8 A . There are two bridging ligands to the metals: a water molecule (or possibly a hydroxide ion) and a carbamylated lysine residue (Lys 169) . The more buried cadmium is surrounded by His 55, His 57, Lys 169, Asp 301, and the bridging water in a trigonal bipyramidal arrangement . The second metal is coordinated in a distorted octahedral geometry by His 201, His 230, Lys 169, the bridging water molecule, and two additional solvents.

Science, 1995 Jun 23, 268(5218), 1758 - 62
Crystal structure of Pseudomonas mevalonii HMG-CoA reductase at 3.0 angstrom resolution; Lawrence CM et al.; The rate-limiting step in cholesterol biosynthesis in mammals is catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, a four-electron oxidoreductase that converts HMG-CoA to mevalonate . The crystal structure of HMG-CoA reductase from Pseudomonas mevalonii was determined at 3.0 angstrom resolution by multiple isomorphous replacement . The structure reveals a tightly bound dimer that brings together at the subunit interface the conserved residues implicated in substrate binding and catalysis . These dimers are packed about a threefold crystallographic axis, forming a hexamer with 23 point group symmetry . Difference Fourier studies reveal the binding sites for the substrates HMG-CoA and reduced or oxidized nicotinamide adenine dinucleotide {NAD(H)} and demonstrate that the active sites are at the dimer interfaces . The HMG-CoA is bound by a domain with an unusual fold, consisting of a central alpha helix surrounded by a triangular set of walls of beta sheets and alpha helices . The NAD(H) is bound by a domain characterized by an antiparallel beta structure that defines a class of dinucleotide-binding domains.

J Biol Chem, 1995 Jun 16, 270(24), 14420 - 9
Molecular cloning of the isoquinoline 1-oxidoreductase genes from Pseudomonas diminuta 7, structural analysis of iorA and iorB, and sequence comparisons with other molybdenum-containing hydroxylases; Lehmann M et al.; The iorA and iorB genes from the isoquinoline-degrading bacterium Pseudomonas diminuta 7, encoding the heterodimeric molybdo-iron-sulfur-protein isoquinoline 1-oxidoreductase, were cloned and sequenced . The deduced amino acid sequences IorA and IorB showed homologies (i) to the small (gamma) and large (alpha) subunits of complex molybdenum-containing hydroxylases (alpha beta gamma/alpha 2 beta 2 gamma 2) possessing a pterin molybdenum cofactor with a monooxo-monosulfido-type molybdenum center, (ii) to the N- and C-terminal regions of aldehyde oxidoreductase from Desulfovibrio gigas, and (iii) to the N- and C-terminal domains of eucaryotic xanthine dehydrogenases, respectively . The closest similarity to IorB was shown by aldehyde dehydrogenase (Adh) from the acetic acid bacterium Acetobacter polyoxogenes . Five conserved domains of IorB were identified by multiple sequence alignments . Whereas IorB and Adh showed an identical sequential arrangement of these conserved domains, in all other molybdenum-containing hydroxylases the relative position of "domain A" differed . IorA contained eight conserved cysteine residues . The amino acid pattern harboring the four cysteine residues proposed to ligate the Fe/S I cluster was homologous to the consensus binding site of bacterial and chloroplast-type {2Fe-2S} ferredoxins, whereas the pattern including the four cysteines assumed to ligate the Fe/S II center showed no similarities to any described {2Fe-2S} binding motif . The N-terminal region of IorB comprised a putative signal peptide similar to typical leader peptides, indicating that isoquinoline 1-oxidoreductase is associated with the cell membrane.

Gene, 1995 Jun 14, 159(1), 73 - 80
Biotechnological and gene therapeutic strategies in cancer treatment; Wels W et al.; New anti-cancer agents are being developed which incorporate cancer-cell-specific recognition functions and are thus able to distinguish between normal and tumor cells . Recognition is dependent on the enhanced expression of antigenic determinants on the surface of tumor cells . The ErbB-2 receptor (ErbB-2R) is overproduced in a high percentage of adenocarcinomas arising in the breast, ovary, lung and stomach, when compared to normal cells . The tumor-enriched expression and extracellular accessibility make this receptor a suitable target for directed tumor therapy . A gene expressing the single-chain antibody molecule (scFv), specific for the extracellular domain of the ErbB-2R, was constructed by joining cDNAs encoding the light- and heavy-chain variable domains of the monoclonal antibody (mAb) FRP5 . This scFv-encoding gene has been used as a targeting domain for two effectors: (i) A recombinant immunotoxin-encoding gene was constructed by adding sequences encoding a modified Pseudomonas aeroginosa exotoxin A (ETA) to the scFv-encoding DNA . (ii) Cytotoxic T-lymphocytes (CTL) with specificity for ErbB-2R-producing tumor cells were generated by retroviral transfer of a chimeric gene which encodes the scFv(FRP5), a hinge region and the zeta-chain of the T-cell receptor (TCR) complex . The bacterially produced recombinant immunotoxin scFv(FRP5)-ETA binds specifically to the ErbB-2R and displays both in vitro and in vivo cytotoxic effects selective for tumor cells producing high levels of the ErbB-2R . Target cells expressing the ErbB-2R gene were lysed in vitro with high specificity by the scFv::hinge::zeta-expressing T-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochim Biophys Acta, 1995 Jun 6, 1256(3), 396 - 402
Lipase from Chromobacterium viscosum: biochemical characterization indicating homology to the lipase from Pseudomonas glumae; Taipa MA et al.; Previous purification of a commercial lipolytic preparation from Chromobacterium viscosum using gel filtration chromatography yielded two enzymatically active fractions, named lipases A and B . Characterization of these fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that lipase A consisted of a high molecular weight aggregate of lipase protein with lipopolysaccharides . This complex could be dissociated by treatment with EDTA-Tris buffer containing the non-ionic detergent n-octyl-beta-D-glucopyranoside and subsequent isoelectric focusing in an agarose gel containing the same detergent . Both lipases A and B revealed a major peak corresponding to an isoelectric point of 7.1 . SDS-PAGE analysis of lipases A and B after purification by gel filtration or by IEF revealed one major protein band of M(r) of 33 K . Determination of N-terminal amino acid sequences confirmed that both fractions A and B contained the same lipase protein . Furthermore, the N-terminal amino acid sequence of the C . viscosum lipase was identical to the one of Pseudomonas glumae lipase.

Ther Immunol, 1995 Jun, 2(3), 137 - 45
Antitumour activity of a melanoma-specific immunotoxin, ME20-LysPE40; Wolff EA et al.; An immunotoxin conjugate has been prepared by linking an internalizing antibody with melanoma selectivity, ME20, with a binding-defective form of Pseudomonas exotoxin A, LysPE40 . ME20-LysPE40 binds to a 105,000 Da cell-surface antigen present on melanoma cells (ME20-M) within twofold of unmodified ME20 and was cytotoxic to two human melanoma cell lines, H3606 and MALME-3M, with EC50 values of 100 and 200 pM, respectively . Immunotoxin treatment, initiated 1 day following subcutaneous implantation of H3606 melanoma cells into mice, prevented outgrowth of tumour xenografts in > 50% of the mice . In contrast, only a modest inhibition in tumour growth was observed if the immunotoxin was administered 5 days after implantation of in vivo passaged H3606 tumour fragments in mice . This study shows that the internalizing monoclonal antibody ME20 IgG can be used for targeting a toxin toward melanoma cells displaying the ME20-M antigen.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1995 Jun, 17(3), 161 - 6
{Cloning and sequencing of the gene PE40, coding for a portion of Pseudomonas exotoxin A}; Zhang M et al.; Gene PE40 coding for a mutant form of Pseudomonas Exotoxin A was devoid of its native cell binding domain, and was amplified from the total DNA of P . aeruginosa PA103 by PCR . After being sequenced, it was cloned into expressional vector pBV220 to get a recombinant plasmid, which will be useful in the research of molecular targeted protein.

Singapore Med J, 1995 Jun, 36(3), 299 - 302
Serodiagnosis of melioidosis in Malaysia; Vadivelu J et al.; Current diagnosis of melioidosis is based on bacterial culture and/or serology which is becoming increasingly useful . An IgM-ELISA using heat-killed whole cells of Pseudomonas pseudomallei was developed and compared with an indirect haemagglutination technique (IHAT) and an indirect immunofluorescent technique(IFAT) . The IgM-ELISA using a P:N ratio of > or = 2 had a sensitivity of 91% and a specificity of 96% . All 3 assays were further used in a seroepidemiological survey amongst different groups of patients and healthy individuals . It was found that the IFAT performed better than the IHAT, detecting antibodies to P . pseudomallei in 6% of diabetics, 5% of pyrexics, 8% of pregnant women and 3% of farmers . For the same groups the IgM-ELISA detected antibodies in 1% of pyrexics, 8% of pregnant women and a further 14% of farmers . The IgM-ELISA was found to be sensitive and useful for the serological diagnosis of acute melioidosis.

Cryobiology, 1995 Jun, 32(3), 247 - 58
Membrane fluidity as a factor in production and stability of bacterial ice nuclei active at high subfreezing temperatures; Lindow SE; Detailed measurements were made of the rate of appearance of bacterial ice nuclei upon cooling of suspensions of Pseudomonas syringae cells and the disappearance of ice nuclei upon warming of the cells before assay for ice nucleation activity . While no substantial change in numbers of ice nuclei active at either -5 or at -9 degrees C was observed in cells that were grown at temperatures lower than 24 degrees C and cooled to 21 degrees C before assay, large increases in -5 but not -9 degrees C ice nuclei were observed in cells grown at temperatures greater than 24 degrees C . Ice nucleation activity of cells subjected to a decrease in temperature before assay increased immediately upon temperature shift, but 8 to 12 min was required before maximum rates of increase in numbers of ice nuclei were observed . The rate of appearance of ice nuclei in cell suspensions incubated at relatively cold temperatures prior to assay was substantially less than those incubated at temperatures approaching 24 degrees C . Cells rapidly lost ice nucleation activity when warmed to above 27 degrees C before assay; the rate of loss of ice nuclei in cells grown at a given temperature increased rapidly as the temperature to which they were warmed before assay increased . Ice nuclei disappeared most rapidly when cells grown at low temperatures were warmed before assay, suggesting that ice nucleus stability was lower in highly fluid membranes . The logarithm of the half-life of ice nuclei in cells was directly related to the concentration of the membrane fluidizing agent, 2-phenethyl alcohol, in which they were suspended.

J Clin Invest, 1995 Jun, 95(6), 2491 - 500
Protease-cleaved iron-transferrin augments oxidant-mediated endothelial cell injury via hydroxyl radical formation; Miller RA et al.; Previous work has shown that the Pseudomonas-derived protease, pseudomonas elastase (PAE), can modify transferrin to form iron complexes capable of catalyzing the formation of hydroxyl radical (.OH) from neutrophil (PMN)-derived superoxide (.O2-) and hydrogen peroxide (H2O2) . As the lung is a major site of Pseudomonas infection, the ability of these iron chelates to augment oxidant-mediated pulmonary artery endothelial cell injury via release of 51Cr from prelabeled cells was examined . Diferrictransferrin previously cleaved with PAE significantly enhanced porcine pulmonary artery endothelial cell monolayer injury from 2.3-6.3 to 15.8-17.0% of maximum, resulting from exposure to H2O2, products of the xanthine/xanthine oxidase reaction, or PMA-stimulated PMNs . Iron associated with transferrin appeared to be responsible for cell injury . Spin trapping and the formation of thiobarbituric acid-reactive 2-deoxyribose oxidation products demonstrated the production of .OH in this system . The addition of catalase, dimethyl thiourea, and the hydrophobic spin trap, alpha-phenyl-n-terbutyl-nitrone, offered significant protection from injury (27.8-58.2%) . Since sites of Pseudomonas infection contain other proteases, the ability of porcine pancreatic elastase and trypsin to substitute for PAE was examined . Results were similar to those observed with PAE . We conclude .OH formation resulting from protease alteration of transferrin may serve as a mechanism of tissue injury at sites of bacterial infection and other processes characterized by increased proteolytic activity.

Rinsho Ketsueki, 1995 Jun, 36(6), 578 - 81
{Targeted toxin therapy in the treatment of leukemia}; Tojo A et al.; A chimeric toxin in which the cell surface binding domain of Pseudomonas exotoxin was replaced with mature human granulocyte colony-stimulating factor (G-CSF) was produced in Escherichia coli, partially purified and tested for its biological activity on a G-CSF-dependent murine myeloid leukemia cell line, NFS60 . This fusion protein, termed as G-CSF-PE40, can displace {125I} G-CSF binding to its receptor . After 48 hrs incubation in the presence of G-CSF, G-CSF-PE40 inhibited protein synthesis and revealed cytotoxicity in NFS60 cells in a concentration dependent manner . The half maximal dose of G-CSF-PE40 for protein synthesis inhibition (ID50) in NFS60 cells was estimated at around 100pM . Additionally, relatively low concentration of G-CSF-PE40 stimulated DNA synthesis in NFS60 cells after 16 hrs' incubation in the absence of G-CSF, suggesting that G-CSF-PE40 also can transduce a transient mitogenic signal . Thus, G-CSF-PE40 may be useful in the selective elimination of myeloid cells expressing G-CSF receptors, especially in combination with chemotherapeutic agents like cytosine arabinoside.

J Biochem (Tokyo), 1995 Jun, 117(6), 1317 - 22
Comprehensive site-directed mutagenesis of L-2-halo acid dehalogenase to probe catalytic amino acid residues; Kurihara T et al.; L-2-Halo acid dehalogenase catalyzes the stereospecific hydrolytic dehalogenation of L-2-halo acids, with inversion of the C2-configuration . Seven L-2-halo acid dehalogenases from various bacterial strains are significantly similar to one another in their amino acid sequences (36-70% identity), and they are supposed to catalyze the reaction through the same mechanism . To identify catalytically important residues, we mutated all the 36 highly conserved charged and polar amino acid residues of L-2-halo acid dehalogenase from Pseudomonas sp . YL, which consists of 232 amino acid residues, by replacement of D by N, E by Q, R by K, and vice versa, S and T by A, Y and W by F, M by L, and H by N . We found that the replacement of D10, K151, S175, D180, R41, S118, T14, Y157, and N177 led to a significant loss in the enzyme activity or an increase in the Km value for the substrate, showing their involvement in the catalysis . The roles of these residues are discussed.

Mol Microbiol, 1995 Jun, 16(5), 977 - 89
Molecular characterization of the eps gene cluster of Pseudomonas solanacearum and its transcriptional regulation at a single promoter; Huang J et al.; Production of EPS I, an unusual exopolysaccharide virulence factor of the phytopathogen Pseudomonas solanacearum, requires the 18 kb eps gene cluster . DNA sequence analysis of the first seven genes of eps (epsAPBCDEF), subcellular localization of their products in maxicells, and phoA fusion analysis showed that: (i) epsA, epsB, epsE, and epsF encode exported or membrane-associated proteins probably involved in polymerization and/or export of EPS I; (ii) epsC and epsD encode soluble enzymes probably involved in synthesis of sugar components of EPS I (N-acetylgalactosaminuronic acid and possibly N-acetyltrideoxygalactose, respectively); and (iii) epsP probably encodes a phosphatase involved in EPS I production in an unknown way . Non-polar insertional mutagenesis showed that most, if not all, of these eps genes are absolutely required for production of EPS I . Using random eps::lacZ fusions and primer extension we located a transcription start site and promoter upstream of epsA . Analysis of a plasmid with this promoter fused to lacZ showed that a 140 bp regulatory region upstream of the eps transcription start site was sufficient for normal regulation of eps transcription by the multicomponent virulence gene regulatory network of P . solanacearum . Deletion of this eps promoter from a plasmid-borne epsAPBCDE::lacZ fusion reduced its expression 10-fold, indicating that this promoter alone is responsible for regulated transcription of an eps operon composed of at least epsAPBCDE . Analysis of genomic and plasmid-borne eps::lacZ fusions suggested that most remaining eps genes are part of this same operon or, and this is less likely, comprise a second co-ordinately regulated eps operon.

Anal Biochem, 1995 May 20, 227(2), 363 - 7
Preparative synthesis of beta-L-malyl-coenzyme A assisted by malyl-coenzyme A synthetase from Pseudomonas AM1; Willibald B et al.; beta-L-Malyl-CoA was synthesized from L-malate, CoA, and ATP in the presence of catalytic amounts of L-malyl-CoA synthetase (thiokinase) from Pseudomona AM1, which had been 50-fold purified by protamine sulfate precipitation, ammonium sulfate precipitation, chromatography on DEAE-cellulose, and affinity chromatography on High Trap Blue in less than 2 days . The homogeneous enzyme was free of L-malyl-CoA lyase and showed 63% homology with succinyl-CoA synthetase from Thermus aquaticus in its N-terminal sequences . Yields of beta-L-{14C}malyl-CoA(1-10 mumol) were 70% before and 65% after purification in 0.1-0.5 mumol portions by high-performance liquid chromatography on a MN Nucleosil 100-7 C8 column . For most biochemical work, the product was partially purified with an overall 45% yield by chromatography on DEAE-Sephacel . The identity of the compound as beta-L-malyl-CoA was verified by chemical and enzymatic tests, and also in comparison with its chemically synthesized counterpart . The enzymatic synthesis, especially of radioactively labeled beta-L-malyl-CoA, is considerably faster, higher in yield, and less problematic than chemical synthesis.

Biochemistry, 1995 May 16, 34(19), 6562 - 72
Ultraviolet spectroscopic evidence for decreased motion of the active site tyrosine residue of delta 5-3-ketosteroid isomerase by steroid binding; Zhao Q et al.; delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni catalyzes the highly efficient conversion of delta 5-3-ketosteroids to delta 4-3-ketosteroids by a stereoselective and intramolecular transfer of the 4 beta-proton to the 6 beta-position . Tyr-14 is the critical general acid and Asp-38 is the general base involved in catalysis . The UV absorption bandwidths of Tyr-14 were much narrower than those of the other two tyrosines (Tyr-55 and Tyr-88) of isomerase or of the N-acetyltyrosine ethyl ester in aqueous solution, suggesting that Tyr-14 is restricted in its mobility . Further immobilization of this residue occurs upon steroid binding . Thus, 5 alpha-estrane-3,17-dione, an A-ring saturated steroid, induces significant narrowing of the tyrosine absorption bands (pi-->pi*) of the main peak (279.5 nm) and the shoulder (285.5 nm) of Tyr-14, with no significant changes in lambda max . No effects of steroid binding were found on the absorption bandwidths of Tyr-55, Tyr-88, or the phenylalanine residues . The ratio of the absorbance (Amax) at the absorption maximum (lambda max) to that at lambda max plus 4 nm (Amax +4) was used as a measure of peak sharpness . Specifically, the ratios of A279.5/A283.5 (main peak) and A285.5/A289.5 (shoulder) of Tyr-14 of the free enzyme at 25 degrees C were 1.25 and 1.89, respectively, and they increased to 1.41 and 2.70, respectively, in the complex . A more precise measurement of the band narrowing from 4.2 to 3.1 nm between the inflection points was obtained from the derivative spectra . The absorption bands of free and steroid-bound isomerase were narrowed significantly by lowering the temperature and were broadened by denaturation, suggesting that the unusual peak-sharpening effects induced by steroid binding arise from the restricted motion of Tyr-14, as well as from more directional hydrogen bonding resulting from the displacement of water molecules from the active site and decreased flexibility of the protein . Larger enthalpy of the sharpening effects was observed for the steroid-bound enzyme (-0.527 +/- 0.016 kcal/mol) than for the free enzyme (-0.250 +/- 0.018 kcal/mol) by lowering the temperature, indicating that interactions of Tyr-14 with its environment, which restrain its motion, are stronger in the steroid-bound enzyme than in the free enzyme . Hydrogen-bonding modes of Tyr-14, mobility of the active site, and protein flexibility are the environmental factors determining the absorption bandwidths of the critical Tyr-14 residue.

FEMS Microbiol Lett, 1995 May 15, 128(3), 301 - 6
Lack of involvement of merT and merP in methylmercury transport in mercury resistant Pseudomonas K-62; Kiyono M et al.; To clarify whether the merT and merP genes play a role in the transport of methylmercury, we constructed a deletion plasmid, pMRD141 which lacked the genes conferring the organomercurial lyase and the mercuric reductase from plasmid, pMRA17 containing the entire broad-spectrum mercury resistance determinants of the 26 kb-plasmid from Pseudomonas K-62 . Plasmid, pMRD141 showed hypersensitivity to Hg2+ but still expressed a normal sensitivity to methylmercury . The mercury-induced hypersensitive cells carrying pMRD141 took up appreciably more 203Hg2+ than the induced resistant cells with pMRA17 and the sensitive cells with cloning vector, Bluescript II SK(+) but no difference in the uptake of CH3(203)Hg2+ among these three strains was found . These results suggested that the merT and merP are only involved in the Hg2+ transport but do not participate in the transport of methylmercury.

Biochim Biophys Acta, 1995 May 11, 1244(1), 17 - 29
Keratanase digestion of keratan sulphates: characterization of large oligosaccharides from the N-acetyllactosamine repeat sequence and from the non-reducing terminal chain caps; Huckerby TN et al.; Keratan sulfate (KS) chains prepared from both bovine tracheal rings and bovine femoral head cartilage were digested with the enzyme keratanase from Pseudomonas species; large repeat-sequence and non-reducing terminal oligosaccharides were fractionated and purified using high-performance ion-exchange chromatography . The main beta-linked pentasulfated hexasaccharide repeat segment, {R6}, GlcNAc(6S)1-1-3Gal(6S)1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S)1-3Gal-ol and the asialo beta-linked capping pentasulfated heptasaccharide, {C7}, Gal1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S) 1-3Gal-ol have been completely characterized by high-field NMR spectroscopy using one- and two-dimensional methods . Partial 1H assignments are summarized for three homologous series of higher oligosaccharides: GlcNAc(6S){1-3Gal(6S)1-4GlcNAc(6S)}2-5(1-3)Gal-0l {R8,R10,R12} Gal1-4GlcNAc(6S){1-3Gal(6S)1-4GlcNAc(6S)}3-5(1-3)Gal-ol {C9,C11,C13} NeuAc alpha 2-3Gal1-4GlcNAc(6S){1-3Gal(6S)1-4GlcNAc(6S)}2-4(1-3)Gal-ol {C8,C10,C12} obtained from keratan sulfate by keratanase cleavage . The first shows that the unsulfated galactose residues within the repeat sequence region of KS may be separated by fully sulfated segments which have a wide distribution of lengths . The others, viz . those with sialylated caps, and the related galactose capped asialo-segments (derived from a KS digestion in which the keratanase also exhibited sialidase activity) represent an homologous series of epitopes in which the first internal unsulfated galactose is located at a position which may be up to five or more fully sulfated N-acetyllactosamine disaccharide repeat units along from the non-reducing terminus of the KS polymer.

Mol Gen Genet, 1995 May 10, 247(3), 323 - 37
Developmental and pathogen-induced activation of an msr gene, str 246C, from tobacco involves multiple regulatory elements; Gough C et al.; A family of genes, the so-called msr genes (multiple stimulus response), has recently been identified on the basis of sequence homology in various plant species . Members of this gene family are thought to be regulated by a number of environmental or developmental stimuli, although it is not known whether any one member responds more specifically to one stimulus, or whether each gene member responds to various environmental stimuli . In this report, we address this question by studying the tobacco msr gene str246C . Using transgenic tobacco plants containing 2.1 kb of 5' flanking DNA sequence from the str246C gene fused to the beta-glucuronidase (GUS) coding region, the complex expression pattern of the str246C promoter has been characterized . Expression of the str246C promoter is strongly and rapidly induced by bacterial, fungal and viral infection and this induction is systemic . Elicitor preparations from phytopathogenic bacteria and fungi activate the str246C promoter to high levels, as do wounding, the application of auxin, auxin and cytokinin, salicylic acid or copper sulfate, indicating the absence of gene specialization within the msr gene family, at least for str246C . In addition, GUS activity was visualized histochemically in root meristematic tissues of tobacco seedlings and is restricted to roots and sepals of mature plants . Finally, analysis of a series of 5' deletions of the str246C promoter-GUS gene fusion in transgenic tobacco plants confirms the involvement of multiple regulatory elements . A region of 83 bp was found to be necessary for induction of promoter activity in response to Pseudomonas solanacearum, while auxin inducibility and root expression are apparently not controlled by this element, since its removal does not abolish either response . An element of the promoter with a negative effect on promoter activation by P . solanacearum was also identified.

J Biochem (Tokyo), 1995 May, 117(5), 1043 - 9
Purification and characterization of the 3-ketosteroid-delta 1-dehydrogenase of Arthrobacter simplex produced in Streptomyces lividans; Choi KP et al.; The 3-ketosteroid-delta 1-dehydrogenase (KS1DH) gene of Arthrobacter simplex IFO12069 cloned in Streptomyces lividans was overexpressed, resulting in production of the enzyme both extracellularly and intracellularly . The enzyme was purified by ammonium sulfate fractionation and chromatographies using DEAE-Toyopearl, Butyl-Toyopearl and Toyopearl HW55S from the supernatant of culture broth and cell-free extracts of S . lividans, and both preparations showed the same characteristics . The N-terminal amino acid sequence of both KS1DHs was M-D-W-A-E-E-Y-D, which coincided with the amino acid sequence deduced from the nucleotide sequence . Thus, the extracellular enzyme may derived from leakage of S . lividans cells during cultivation rather than secretion by processing of the signal sequence . The molecular weight of the enzyme was about 55,000, identical with the size deduced from the nucleotide sequence (M(r) 54,329) . The optimum conditions for its activity were pH 10.0 and 40 degrees C . The enzyme catalyzed the conversion of several 3-keto-steroids, but those containing 11 alpha- or 11 beta-hydroxyl group were converted at low rates . The amino acid sequence of KS1DH from A . simplex is similar to those of KS1DH of Pseudomonas testosteroni and fumarate reductase from Shewanella putrefaciens.

Curr Microbiol, 1995 May, 30(5), 291 - 8
Purification, characterization and antitumor activity of L-asparaginase isolated from Pseudomonas stutzeri MB-405; Manna S et al.; An L-asparaginase produced by Pseudomonas stutzeri MB-405 was isolated and characterized . After initial ammonium sulfate fractionation, the enzyme was purified by consecutive column chromatography on Sephadex G-100, Ca-hydroxylapatite, and DEAE-Sephadex A-50 . The 665.5-fold purified enzyme thus obtained has the specific activity of 732.3 units mg protein-1 with an overall recovery of 27.2% . The apparent M(r) of the enzyme under nondenaturing and denaturing conditions was 34 kDa and 33 kDa respectively, and the isoelectric point was 6.38 +/- 0.02 . It displayed optimum activity at pH 9.0 and 37 degrees C . The enzyme was very specific for L-asparagine and did not hydrolyze L-glutaminate . The Km of the L-asparaginase was found to be 1.45 x 10(-4) M towards L-asparagine and was competitively inhibited by 5-diazo-4-oxo-L- norvaline (DONV) with a Ki of 0.03 mM . Metal ions such as Mn2+, Zn2+, Hg2+, Fe3+, Ni2+, and Cd2+ potentially inhibited the enzyme activity . The activity was enhanced in the presence of thiol-protecting reagents such as DTT, 2-ME, and glutathione (reduced), but inhibited by PCMB and iodoacetamide . The tumor inhibition study with Dalton's lymphoma tumor cells in vivo indicated that this enzyme possesses antitumor properties.

J Bacteriol, 1995 May, 177(10), 2615 - 21
Desaturation, dioxygenation, and monooxygenation reactions catalyzed by naphthalene dioxygenase from Pseudomonas sp . strain 9816-4; Gibson DT et al.; The stereospecific oxidation of indan and indene was examined with mutant and recombinant strains expressing naphthalene dioxygenase of Pseudomonas sp . strain 9816-4 . Pseudomonas sp . strain 9816/11 and Escherichia coli JM109(DE3){pDTG141} oxidized indan to (+)-(1S)-indanol, (+)-cis-(1R,2S)-indandiol, (+)-(1S)-indenol, and 1-indanone . The same strains oxidized indene to (+)-cis-(1R,2S)-indandiol and (+)-(1S)-indenol . Purified naphthalene dioxygenase oxidized indan to the same four products formed by strains 9816/11 and JM109(DE3){pDTG141} . In addition, indene was identified as an intermediate in indan oxidation . The major products formed from indene by purified naphthalene dioxygenase were (+)-(1S)-indenol and (+)-(1R,2S)-indandiol . The results show that naphthalene dioxygenase catalyzes the enantiospecific monooxygenation of indan to (+)-(1S)-indanol and the desaturation of indan to indene, which then serves as a substrate for the formation of (+)-(1R,2S)-indandiol and (+)-(1S)-indenol . The relationship of the desaturase, monooxygenase, and dioxygenase activities of naphthalene dioxygenase is discussed with reference to reactions catalyzed by toluene dioxygenase, plant desaturases, cytochrome P-450, methane monooxygenase, and other bacterial monooxygenases.

Am J Kidney Dis, 1995 May, 25(5), 769 - 74
Pseudomonas peritonitis in peritoneal dialysis patients: the Network #9 Peritonitis Study; Bunke M et al.; To determine risk factors for the development of Pseudomonas peritonitis (PsP) and outcomes of PsP, the authors compared peritoneal dialysis patients who developed PsP with peritoneal dialysis patients who developed non-Pseudomonas bacterial peritonitis (non-PsP) . The authors also sought to determine if there were differences in patients who had resolution of PsP compared with those patients whose PsP did not resolve . The data were derived from the prospective Tristate Renal Network Peritonitis and Catheter Survival Study . Resolution in this study was defined as clearing of peritoneal dialysate on visual inspection, with up to three courses of antibiotic therapy allowed . Catheter removal, switch to hemodialysis, or death were outcomes that were considered separately from resolution because of the study design . There were 31 cases of PsP in 28 patients and 886 cases of non-PsP identified in 667 adult patients . There were no differences in race, gender, age, or incidence of diabetes between the groups . The PsP group had a 25% incidence of previous exposure to immunosuppressive agents, whereas it was 10.6% in the non-PsP group (P = 0.028) . PsP infections were more frequently associated with concomitant exit and tunnel infections, higher hospitalization rates, increased incidence of catheter loss, switch to hemodialysis, and a worse rate of resolution when compared with non-PsP (all, P < 0.05) . Logistic regression could not identify patients at increased risk of PsP . PsP resolved with antibiotic therapy only in 10 of 31 episodes.(ABSTRACT TRUNCATED AT 250 WORDS)

Cancer Res, 1995 May 1, 55(9), 1820 - 3
Decreased expression and function of alpha-2 macroglobulin receptor/low density lipoprotein receptor-related protein in photodynamic therapy-resistant mouse tumor cells; Luna MC et al.; Parental and photodynamic therapy (PDT)-resistant mouse, radiation-induced fibrosarcoma cell lines were evaluated using mRNA differential display in an attempt to identify unique transcripts . We detected one transcript that was consistently present in the parental cells but absent in PDT-resistant cells . The transcript was cloned, sequenced, and identified as alpha-2 macroglobulin receptor/low density lipoprotein receptor-related protein (alpha-2 MR/LRP) . Northern and Western immunoblot analysis confirmed that receptor expression was present in the parental cell line but barely detectable in PDT-resistant cells . Functionality of the receptor was evaluated by exposing cells to Pseudomonas exotoxin A . alpha-2 MR/LRP is responsible for Pseudomonas exotoxin A internalization, and only the parental cells exhibited toxin-mediated cytotoxicity . The binding and endocytosis of activated alpha-2 macroglobulin and lipoproteins by alpha-2 MR/LRP are consistent with modulating uptake and localization of photosensitizers . Our results demonstrate that PDT-resistant murine tumor cells exhibit minimal alpha-2 MR/LRP activity and suggest that this receptor plays a role in PDT sensitivity by modulating photosensitizer uptake and/or subcellular localization.

Mikrobiol Z, 1995 May-Jun, 57(3), 84 - 97
{The genetic aspects of the study of Pseudomonas syringae bacteria}; Perepnikhatka VI et al.; The work is a review of the state-of-art of molecular-genetic examination of bacteria P . syringae . The questions concerning new approaches to the study of determinants of pathogenicity, endogenic plasmids, toxins, resistance to antibiotics, avirulence, genes and insertion sequence elements of P . syringae are stated.

Mol Plant Microbe Interact, 1995 May-Jun, 8(3), 444 - 53
The avrRpm1 gene of Pseudomonas syringae pv . maculicola is required for virulence on Arabidopsis; Ritter C et al.; We demonstrate that the avirulence gene avrRpm1, isolated from Pseudomonas syringae pv . maculicola strain Psm M2 via interaction with the Arabidopsis resistance gene RPM1, is also required for maximal virulence on this host . Two avrRpm1::Tn3-Spice marker-exchange mutants do not elicit a hypersensitive reaction on RPM1-containing Arabidopsis accessions Col-0 and Oy-0 . Surprisingly, these mutants neither generate disease symptoms, nor grow in planta, after inoculation onto susceptible accessions Nd-0, Fe-1, and Mt-0 . These deficiencies can be corrected in a merodiploid containing a wild-type avrRpm1 allele, and are not observed following gene-replacement with avrRpm1::Tn3-Spice alleles containing insertions just beyond the 3' terminus of the avirulence gene open reading frame . AvrRpm1 mRNA is expressed in low, but detectable amounts, in rich media . Induced accumulation of transcript is observed 3 h after shift to minimal media, and an avrRpm1::Tn3-Spice marker-exchanged reporter gene reaches maximal induction 30 min after shift . AvrRpm1 transcription starts 5 base-pairs 3' of the putative regulatory "hrp-box" cis-element found upstream of many P . syringae avr and hrp genes . Transcriptional induction of the marker-exchanged reporter gene in minimal media is enhanced by a carbon source . Induction in planta is the same in either resistant or susceptible Arabidopsis accessions, and is unaffected by the presence or absence of wild-type avrRpm1 . As previously observed for many other P . syringae avr genes, transcriptional regulation of avrRpm1 in minimal media is dependent on hrpL and hrpS.

Appl Environ Microbiol, 1995 May, 61(5), 2004 - 6
A unique endoglucanase-encoding gene cloned from the phytopathogenic fungus Macrophomina phaseolina; Wang H et al.; The deduced amino acid sequence derived from a Macrophomina phaseolina beta-1,4-endoglucanase-encoding gene revealed 48% identity (over 119 amino acids) with egl1 from the phytopathogen Pseudomonas solanacearum . Its similarity to saprophyte endoglucanases was not significant . Its minimum substrate size, unlike that of any known saprophyte endoglucanase, was cellopentaose . The unique characteristics of M . phaseolina egl1-encoded endoglucanase suggest that it is phytopathogen specific.

Clin Infect Dis, 1995 May, 20(5), 1327 - 32
Shewanella (Pseudomonas) putrefaciens bacteremia; Brink AJ et al.; Shewanella (Pseudomonas) putrefaciens is a rare pathogen in humans, and to our knowledge only 13 cases of S . putrefaciens bacteremia have ever been reported in the literature . In this retrospective study we describe 28 cases of S . putrefaciens bacteremia: 16 in premature and 1-day-old neonates, 9 in adults, and 3 in children younger than 1 year of age . All the babies presented with respiratory distress and/or pneumonia . Six of the adults had associated traumatic lesions of the lower extremity, and of the eight patients who died of sepsis, six were neutropenic . Polymicrobial bacteremia was seen in 18 cases . Three syndromes of bacteremic infection with S . putrefaciens appear to exist: the first is associated with prematurity and congenital pneumonia; the second with ulceration of the lower extremities; and the third (which is more fulminant), with an underlying debility . The findings in these 28 cases confirm the fact that S . putrefaciens can invade the bloodstream; however, the presence of concurrent bacteremia makes it difficult to determine the pathogenic role of this organism in septicemia.

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