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-tr