Pseudomonas

Mol Immunol, 1993 Mar, 30(4), 379 - 86
Insertion of constant region domains of human IgG1 into CD4-PE40 increases its plasma half-life; Batra JK et al.; CD4-PE40 is a recombinant toxin containing the binding domain of CD4 and a mutant form of Pseudomonas exotoxin A from which the cell binding domain has been removed . To increase the serum half-life of CD4-PE40, we have inserted various portions of the constant domain of human IgG1 into CD4-PE40 . The constructs made include CD4-CH2-PE40, CD4-CH3-PE40, CD4-CH1-CH2-PE40 and CD4-CH2-CH3-PE40 . The fusion proteins were expressed and purified from E . coli . Insertion of various domains from the constant region of IgG1 did not alter the cytotoxic activity of CD4-PE40; all these molecules were equally cytotoxic to cells expressing gp120 on their surface . However, there was a marked increase in the serum mean residence time of CD4-CH2-PE40 which was 115 min as compared to 47 min for CD4-PE40 . Insertion of other domains also increased the half-life of CD4-PE40, however, CD4-CH2-PE40 was found to have the longest mean residence time in the circulation . One possible explanation for the increase in plasma half-life is diminished susceptibility of proteins to proteolysis . It was found that CD4-CH2-PE40 was much more resistant to proteolysis by trypsin than CD4-PE40 . We proposed that insertion of the CH2 domain into CD4-PE40 covers up the protease sensitive sites in the molecule, thereby making the molecule less susceptible to degradation . The increase in size and reduced sensitivity to proteases could both be responsible for the increased plasma half-life of CD4-CH2-PE40.

J Bacteriol, 1993 Mar, 175(6), 1831 - 7
Cloning and characterization of Pseudomonas sp . strain DNT genes for 2,4-dinitrotoluene degradation; Suen WC et al.; The degradation of 2,4-dinitrotoluene (DNT) by Pseudomonas sp . strain DNT is initiated by a dioxygenase attack to yield 4-methyl-5-nitrocatechol (MNC) and nitrite . Subsequent oxidation of MNC by a monooxygenase results in the removal of the second molecule of nitrite, and further enzymatic reactions lead to ring fission . Initial studies on the molecular basis of DNT degradation in this strain revealed the presence of three plasmids . Mitomycin-derived mutants deficient in either DNT dioxygenase only or DNT dioxygenase and MNC monooxygenase were isolated . Plasmid profiles of mutant strains suggested that the mutations resulted from deletions in the largest plasmid . Total plasmid DNA partially digested by EcoRI was cloned into a broad-host-range cosmid vector, pCP13 . Recombinant clones containing genes encoding DNT dioxygenase, MNC monooxygenase, and 2,4,5-trihydroxytoluene oxygenase were characterized by identification of reaction products and the ability to complement mutants . Subcloning analysis suggests that the DNT dioxygenase is a multicomponent enzyme system and that the genes for the DNT pathway are organized in at least three different operons.

J Bacteriol, 1993 Mar, 175(6), 1656 - 64
A two-component regulatory system required for copper-inducible expression of the copper resistance operon of Pseudomonas syringae; Mills SD et al.; Specific induction of the copper resistance operon (cop) promoter from Pseudomonas syringae was measured by beta-galactosidase production from a cop promoter-lacZ fusion . Induction of the cop promoter in P . syringae pv . syringae required trans-acting factors from copper resistance plasmid pPT23D, from which cop was originally cloned . Tn5 mutagenesis of pPT23D was used to localize two complementation groups immediately downstream from copABCD . Cloning and sequencing of the DNA in this region revealed two genes, copR and copS, expressed in the same orientation as the cop operon but from a separate constitutive promoter . The amino acid sequence deduced from these genes showed distinct similarities to known two-component regulatory systems, including PhoB-PhoR and OmpR-EnvZ . In addition, CopR showed strong similarity to copper resistance activator protein PcoR from Escherichia coli . Functional chromosomal homologs to copRS activated the cop promoter, in a copper-inducible manner, in copper-resistant or -sensitive strains of P . syringae pv . tomato and other Pseudomonas species . This implies that copper-inducible gene regulation is associated with a common chromosomally encoded function, as well as plasmid-borne copper resistance, in Pseudomonas spp.

J Bacteriol, 1993 Mar, 175(6), 1621 - 8
Metabolism of 2,2'-dihydroxybiphenyl by Pseudomonas sp . strain HBP1: production and consumption of 2,2',3-trihydroxybiphenyl; Kohler HP et al.; Cells of Pseudomonas sp . strain HBP1 grown on 2-hydroxy- or 2,2'-dihydroxybiphenyl contain NADH-dependent monooxygenase activity that hydroxylates 2,2'-dihydroxybiphenyl . The product of this reaction was identified as 2,2',3-trihydroxybiphenyl by 1H nuclear magnetic resonance and mass spectrometry . Furthermore, the monooxygenase activity also hydroxylates 2,2',3-trihydroxybiphenyl at the C-3' position, yielding 2,2',3,3'-tetrahydroxybiphenyl as a product . An estradiol ring cleavage dioxygenase activity that acts on both 2,2',3-tri- and 2,2',3,3'-tetrahydroxybiphenyl was partially purified . Both substrates yielded yellow meta-cleavage compounds that were identified as 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid and 2-hydroxy-6-(2,3-dihydroxyphenyl)-6-oxo-2,4-hexadienoic acid, respectively, by gas chromatography-mass spectrometry analysis of their respective trimethylsilyl derivatives . The meta-cleavage products were not stable in aqueous incubation mixtures but gave rise to their cyclization products, 3-(chroman-4-on-2-yl)pyruvate and 3-(8-hydroxychroman-4-on-2-yl)pyruvate, respectively . In contrast to the meta-cleavage compounds, which were turned over to salicylic acid and 2,3-dihydroxybenzoic acid, the cyclization products are not substrates to the meta-cleavage product hydrolase activity . NADH-dependent salicylate monooxygenase activity catalyzed the conversions of salicylic acid and 2,3-dihydroxybenzoic acid to catechol and pyrogallol, respectively . The partially purified estradiol ring cleavage dioxygenase activity that acted on the hydroxybiphenyls also produced 2-hydroxymuconic semialdehyde and 2-hydroxymuconic acid from catechol and pyrogallol, respectively.

J Bacteriol, 1993 Mar, 175(6), 1596 - 604
Cloning and nucleotide sequence of the gene encoding the positive regulator (DmpR) of the phenol catabolic pathway encoded by pVI150 and identification of DmpR as a member of the NtrC family of transcriptional activators; Shingler V et al.; The catabolic plasmid pVI150 of Pseudomonas sp . strain CF600 encodes all the genetic information required for the regulated metabolism of phenol and some of its methyl-substituted derivatives . The structural dmp genes of the pathway are clustered in a single operon that lies just downstream of a -24 TGGC, -12 TTGC nif/ntr-like promoter sequence . Promoters of this class are recognized by a minor form of RNA polymerase utilizing sigma 54 (NtrA, RpoN) . Primer extension analysis demonstrated that the dmp operon transcript initiates downstream of the -24, -12 promoter . Transposon insertion mutants, specifically defective in the regulation of the dmp operon, were isolated, and complementation of a phenol-utilization regulatory mutant was used to identify the regulatory locus, dmpR . The 67-kDa dmpR gene product alone was shown to be sufficient for activation of transcription from the dmp operon promoter . Nucleotide sequence determination revealed that DmpR belongs to the NtrC family of transcriptional activators that regulate transcription from -24, -12 promoters . The deduced amino acid sequence of DmpR has high homology (40 to 67% identity) with the central and carboxy-terminal regions of these activators, which are believed to be involved in the interaction with the sigma 54 RNA polymerase and in DNA binding, respectively . The amino-terminal region of DmpR was found to share 64% identity with the amino-terminal region of XylR, which is also a member of this family of activators . This region has been implicated in effector recognition of aromatic compounds that is required for the regulatory activity of XylR.

Plant J, 1993 Mar, 3(3), 457 - 62
Expression of the alpha-thionin gene from barley in tobacco confers enhanced resistance to bacterial pathogens; Carmona MJ et al.; Thionins are cysteine-rich, 5 kDa polypeptides which are toxic to plant pathogens in vitro . Expression of the gene encoding alpha-thionin from barley endosperm, under the 35S promoter from cauliflower mosaic virus, conferred to transgenic tobacco enhanced resistance to the bacterial plant pathogens Pseudomonas syringae pv . tabaci 153 and P . syringae pv . syringae . The barley alpha-thionin gene, which has two introns, was correctly spliced in tobacco . The alpha-thionin in transgenic plants had the expected mobility in the gradient, when separated by high-performance liquid chromatography, reacted with monospecific antibodies and showed the expected antibiotic properties in vitro.

FEMS Microbiol Lett, 1993 Mar 1, 107(2-3), 205 - 10
Restriction fragment length polymorphism analysis of Fusobacterium necrophorum using a novel repeat DNA sequence and a 16S rRNA gene probe; Hodgson AL et al.; A repeated DNA sequence was isolated from Fusobacterium necrophorum biotype AB, strain FnS1 . The repeated sequence shared considerable homology with the transposase gene from the Pseudomonas syringiae insertion sequence IS801 . The repeat sequence was used together with a 16S ribosomal RNA gene probe to type F . necrophorum isolates using restriction fragment length polymorphisms . The probes revealed differences between several clinical isolates and will be useful tools to study the epidemiology of ovine foot abscess and other diseases caused by F . necrophorum.

Bioconjug Chem, 1993 Mar-Apr, 4(2), 112 - 20
Single-chain immunotoxin fusions between anti-Tac and Pseudomonas exotoxin: relative importance of the two toxin disulfide bonds; Kreitman RJ et al.; Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin in which the variable heavy and light domains of the anti-IL2 receptor antibody, anti-Tac, are connected to each other by a peptide linker and then fused to PE40, a truncated form of Pseudomonas exotoxin (PE) . This fusion protein has four disulfide bonds: one in each of the two variables domains, one in domain II (Cys 265-287), and one in domain Ib (Cys 372-379) of PE . To study the importance of the disulfide bonds of the toxin to the activity of single-chain immunotoxins, we constructed mutants in which either the cysteines in the toxin were changed to alanines or the amino acids 365-380 of PE were deleted . We began this study with anti-Tac(Fv)-PE40 and a more active variant, anti-Tac(Fv)-PE40KDEL, in which the carbonyl terminus is changed from REDLK to KDEL . From these proteins we made anti-Tac(Fv)-PE40(4)A and anti-Tac(Fv)-PE40KDEL4A, respectively, by converting cysteins at amino acids 265, 287, 372, and 379 of PE to alanines . This change resulted in a 20-100-fold loss of activity toward human target cells, but no significant change in binding affinity to p55 . To determine the importance of the second toxin disulfide bond, we removed amino acids 365-380 from anti-Tac(Fv)-PE40, anti-Tac(Fv)-PE40KDEL, and anti-Tac(Fv)-PE40KDEL4A, resulting in anti-Tac(Fv)-PE38, anti-Tac(Fv)-PE38KDEL, and anti-Tac(Fv)-PE38KDEL2A, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Infect Control Hosp Epidemiol, 1993 Mar, 14(3), 127 - 30
Case-control and vector studies of nosocomial acquisition of Pseudomonas cepacia in adult patients with cystic fibrosis; Burdge DR et al.; OBJECTIVE: To examine factors associated with nosocomial acquisition of Pseudomonas cepacia in adult patients with cystic fibrosis . DESIGN: A retrospective case-control study of 5 patients with nosocomial acquisition of P cepacia versus 20 matched controls who failed to develop P cepacia infection . Selective handwashing, air sampling, and respiratory equipment sampling also were performed . SETTING: A university hospital providing tertiary care to 95 adult cystic fibrosis patients . PATIENTS: All patients are adults with known cystic fibrosis . Case definition required multiple negative sputum cultures for P cepacia prior to and during admission, with a positive sputum culture prior to discharge . Controls had negative sputum cultures for P cepacia prior to and throughout hospitalization . Controls were matched for age, gender, disease severity, and frequency of hospitalizations . RESULTS: Factors associated with increased risk of nosocomial acquisition of P cepacia included receiving humidifier or nebulized treatments (60% versus 5%, p = .016, odds ratio = 28.5, 95% confidence interval = 1.93 to 420.58) . Factors without significance included ward, room, teaching versus nonteaching status, use of steroids, sharing a hospital room with another cystic fibrosis patient, antibiotic use, presence of portocath in situ, or socializing with another individual with cystic fibrosis known to be P cepacia-positive . Air sampling studies failed to demonstrate aerosolization of P cepacia by coughing cystic fibrosis patients over a 1-hour sampling time . Handwashing studies failed to demonstrate P cepacia on hands of cystic fibrosis patients, nurses, or physiotherapists (before or after physiotherapy) . Reservoirs from nebulizers consistently grew P cepacia following therapy . CONCLUSIONS: Respiratory equipment may be an important source of nosocomial acquisition of P cepacia in adult cystic fibrosis patients.

J Clin Microbiol, 1993 Mar, 31(3), 533 - 9
Molecular typing of Pseudomonas pseudomallei: restriction fragment length polymorphisms of rRNA genes; Lew AE et al.; The aim of this study was to develop a typing scheme for Pseudomonas pseudomallei by comparison of patterns of restriction fragment length polymorphisms in rRNA genes (ribotyping) . BamHI restriction digests of 100 isolates from various animal (34), human (58), and environmental (6) sources, including six reference strains, were hybridized to Escherichia coli 16S and 23S rRNAs . A chemiluminescent labelling and detection system was used to visualize bands . On the basis of patterns, the strains were classified into 22 different groups, with the largest containing 29 isolates . While most of the ribotypes were not exclusive to a particular source, some ribotypes were restricted to a particular geographic area or to either a human or a particular animal species . Application of the typing scheme to isolates of four independent outbreaks among animals showed that certain ribotypes predominated . The study demonstrated ribotyping to be a useful tool in epidemiological investigations of melioidosis.

Clin Infect Dis, 1993 Mar, 16(3), 407 - 11
Outbreak of Pseudomonas cepacia bacteremia in oncology patients; Pegues DA et al.; In 1991, an outbreak of Pseudomonas cepacia bacteremia (PCB) occurred among patients at an oncology clinic in Alabama . A case-patient was defined as any patient at Alabama Oncology Hematology Associates (AOHA) who had at least one blood culture positive for P . cepacia from 7 August through 31 October . Fourteen case-patients were identified; all required hospitalization (median duration, 17 days), but none died of PCB . A cohort study assessing risk factors for PCB focused on all patients who had been treated on the 8 days when case-patients had last visited AOHA during the period 7-21 August . Only patients with central venous catheters developed PCB (P < .001) . Among patients with central venous catheters, PCB occurred only after visits to AOHA at which the catheters were flushed with heparin solution in the AOHA laboratory rather than in the treatment area (P < .001) . P . cepacia was cultured from the only intravenous fluid bag used to prepare heparin flush solution in the laboratory during the interval 7-21 August . All outbreak-associated isolates of P . cepacia had an identical DNA ribotype pattern . These findings emphasize the importance of avoiding multiple use of single-use solutions, especially for high-risk patients with long-term indwelling central venous catheters.

Microb Releases, 1993 Mar, 1(4), 209 - 16
Construction and application of chromosomally integrated lac-lux gene markers to monitor the fate of a 2,4-dichlorophenoxyacetic acid-degrading bacterium in contaminated soils; Masson L et al.; A reporter gene system, containing luxAB and lacZY, was constructed and integrated, using Tn7 transposition, into the chromosome of a 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading soil bacterium, Pseudomonas cepacia (BRI6001), to monitor its fate when introduced into soil microcosms . The genes were stably maintained in the modified strain of BRI6001, BRI6001L, for more than 300 generations in the absence of selection pressure, and had no apparent effects on biochemical or physiological properties . BRI6001L was easily and rapidly identified as light-emitting blue colonies on 2,4-D medium containing XGal (5-bromo-4-chloro-indolyl-beta-D-galacto-pyranoside) in the presence of n-decanal . Survival rates of BRI6001L introduced into non-sterile soil microcosms were substrate- and contaminant-dependent . The decrease in population density was lowest in a 2,4-D-amended agricultural soil, and highest in a wood-treatment facility soil contaminated with pentachlorophenol, creosote and heavy metals . A viable cell density as low as 10 cfu g-1 was detected in soil microcosms . The biochemical and growth properties of BRI6001 and BRI6001L, and their behaviour when introduced into soil microcosms indicates that BRI6001L can be used as a reliable model to predict the fate of BRI6001 when used to bioaugment contaminated soil.

Biochemistry, 1993 Feb 23, 32(7), 1816 - 24
Environments and mechanistic roles of the tyrosine residues of delta 5-3-ketosteroid isomerase; Li YK et al.; Delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni converts delta 5-3-ketosteroids to delta 4-3-ketosteroids by a stereoselective and conservative transfer of the 4 beta-proton to the 6 beta-position . The 10(9.5)-fold enzymatic rate acceleration can be attributed to a concerted rate-limiting enolization in which Tyr-14 and Asp-38, positioned orthogonally, act as general acid and base, respectively . The pKa value of the phenolic hydroxyl group of Tyr-14 of the Y55F/Y88F double mutant is 11.6 +/- 0.2 by UV titration . However, the fluorescence titration of Tyr-14 shows biphasic sigmoidal behavior with apparent pKa values of 9.5 and 11.5 . This suggests the assistance of a basic residue at the active site, possibly a lysine or tyrosine residue . Mutation of each of the four lysine residues K119L, K108Q, K92Q, and K60Q lowered specific activities only slightly to between 43% and 98% of the wild-type enzyme . Similarly, mutations of Tyr-55, Tyr-88, or both to phenylalanine led to only 2-4-fold reductions in catalytic activity . These findings suggest that despite the enormous difference between the pKa value of Tyr-14 (11.6) and that of the 3-carbonyl group of the steroid (ca . pKa-7), the reaction may rely on the concerted participation of Tyr-14 and Asp-38 only . The apparent pKa value of 9.5 in the fluorescence titration of Tyr-14 and in kinetic measurements probably results from conformational changes of the enzyme . The unusually high pKa value of Tyr-14 of 11.6 +/- 0.2 was used to estimate a local dielectric constant of 18 +/- 2 near this residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Cancer Res, 1993 Feb 15, 53(4), 819 - 25
Pseudomonas exotoxin-based immunotoxins containing the antibody LL2 or LL2-Fab' induce regression of subcutaneous human B-cell lymphoma in mice; Kreitman RJ et al.; We have produced immunotoxins using LL2, a monoclonal antibody which binds to human B-cell lymphomas and which, in a radioiodinated form, induced responses in lymphoma patients (D.M . Goldberg et al., J . Clin . Oncol., 9: 548-564, 1991) . We have coupled LL2 to Lys-PE38KDEL, a derivative of Pseudomonas exotoxin (PE) which does not bind to the PE receptor . LL2-PE38KDEL was cytotoxic toward several Burkitt's lymphoma lines, with 50% inhibitory concentration values ranging from 2 to 6 ng/ml (10-30 pM) . Another immunotoxin, LL2-Fab'-PE38KDEL, was produced by chemically coupling the Fab' fragment of LL2 to Lys-PE38KDEL . LL2-Fab'-PE38KDEL also was cytotoxic toward the Burkitt's cells, with a 50% inhibitory concentration of 1-2 ng/ml (13-24 PM) . The antibody LL2 alone had no cytotoxicity toward the malignant cells, and excess LL2 prevented the cytotoxicity of LL2-PE38KDEL and LL2-Fab'-PE38KDEL . Control immunotoxins UPC-10-PE38KDEL and Mu-9-Fab'-PE38KDEL were not cytotoxic . LL2-PE38KDEL and LL2-Fab'-PE38KDEL bound to cells with 50% and 17% of the affinity of LL2, respectively . Both immunotoxins, but not UPC-10-PE38KDEL, prevented the development of CA-46 tumors in nude mice . LL2-PE38KDEL and LL2-Fab'-PE38KDEL, but not the control immunotoxins, led to complete regressions of measurable s.c . CA-46 tumors in nude mice, when given at 50% and 35% of the 50% lethal dose, respectively . LL2 alone significantly retarded the growth of CA-46 tumors but did not cause complete tumor regressions . Immunotoxins containing derivatives of Pseudomonas exotoxin can be targeted to human B-cell lymphoma and merit further study as potential therapeutic agents.

J Biol Chem, 1993 Feb 5, 268(4), 2590 - 4
Expression of mouse furin in a Chinese hamster cell resistant to Pseudomonas exotoxin A and viruses complements the genetic lesion; Moehring JM et al.; RPE.40 is a strain of mutated CHO-K1 cells with elevated resistance to Pseudomonas exotoxin A, Sindbis virus, and Newcastle disease virus . Virus resistance is due to an inability to cleave precursor viral membrane glycoproteins and produce infectious virions . Transfection of RPE.40 cells with cDNA for mouse furin causes them to lose all resistance and become as sensitive as wild-type cells to the toxin and viruses . Transfection of RPE.40 cells with cDNA for the related yeast protease Kex2 reduces their resistance to the toxin and viruses, but does not completely eliminate it.

Int J Epidemiol, 1993 Feb, 22(1), 88 - 95
Epidemiology of sudden unexpected death syndrome among Thai migrant workers in Singapore; Goh KT et al.; A total of 235 cases of sudden unexpected death syndrome (SUDS) among apparently healthy male Thai migrant workers in Singapore were reported between 1982 and 1990 . Most of the deaths occurred during sleep and 13% were not sleep-related . The median age at the time of death was 33 years and the median interval between arrival and death was 8 months . These deaths occurred singly and sporadically throughout the year . Post-mortem examination revealed few abnormal findings except for haemorrhagic congestion or oedema of the lungs . There were moderate to severe intra-alveolar haemorrhages with some evidence of myocarditis or pneumonitis . Preliminary findings of serial sections of the hearts indicate evidence of anomalies in the cardiac conduction system . Epidemiological investigations showed that a family history of similar deaths and serological evidence of current or recent infection with Pseudomonas pseudomallei were significantly associated with SUDS . Extensive biochemical and toxicological investigations were inconclusive . There was no evidence of chronic deficiency in thiamine or potassium among the healthy Thai workers living and working in the same conditions as the cases, and no significant abnormalities were detected on electrocardiographic examination . As these migrant workers experienced various psychosocial problems which could stem from maladjustment to an urban environment, separation from the family, burden of debts and long hours of work, stress could be a precipitating factor for SUDS.

Burns, 1993 Feb, 19(1), 12 - 6
Evidence for Kupffer cell activation by burn injury and Pseudomonas exotoxin A; Dong YL et al.; Postburn metabolic and immunological alterations may in part be due to translocation of gut exotoxin and endotoxin, which can result in tumour necrosis factor (TNF) and prostaglandin E (PGE) production by macrophages . We evaluated the effect of burn injury, plus exotoxin and endotoxin on TNF-alpha and PGE production by Kupffer cells, and peritoneal macrophages . Adult Wistar rats underwent 30 per cent TBSA burn or sham burn . Kupffer cells were harvested from rat livers and peritoneal macrophages from the abdominal cavity 24 h postburn . They were cultured overnight at 1 x 10(6) cells/ml and stimulated with saline, 5 micrograms/ml of Pseud . aeruginosa Exotoxin A (Exo-A), 5 micrograms/ml of Pseud . aeruginosa Endotoxin (Endo), Exo-A + Endo, or Exo-A + Endo + the PGE derivative 16,16 dimethyl-PGE (dPGE) (10 micrograms/ml) . The supernatants were harvested after 4, 24 and 48 h of culture and assayed for TNF-alpha and PGE . Results showed that burn injury induced an increase in TNF-alpha and PGE production by Kupffer cells stimulated with Exo-A, Endo, and both Exo-A + Endo (P < 0.05) . The release of TNF-alpha by Kupffer cells was downregulated by exogenous PGE (P < 0.05) . The increased TNF-alpha production was inversely related to PGE levels . In conclusion, both burn injury and Exo-A potentiate the responsiveness of Kupffer cells and peritoneal macrophages to endotoxin as measured by the rate of production of TNF-alpha and PGE . PGE may locally downregulate the immune response by limiting Kupffer cells' and peritoneal macrophages' TNF-alpha production.

Appl Environ Microbiol, 1993 Feb, 59(2), 528 - 35
Degradation of 2-chloroallylalcohol by a Pseudomonas sp; van der Waarde JJ et al.; Three Pseudomonas strains capable of utilizing 2-chloroallylalcohol (2-chloropropenol) as the sole carbon source for growth were isolated from soil . The fastest growth was observed with strain JD2, with a generation time of 3.6 h . Degradation of 2-chloroallylalcohol was accompanied by complete dehalogenation . Chloroallylalcohols that did not support growth were dechlorinated by resting cells; the dechlorination level was highest if an alpha-chlorine substituent was present . Crude extracts of strain JD2 contained inducible alcohol dehydrogenase activity that oxidized mono- and dichloroallylalcohols but not trichloroallylalcohol . The enzyme used phenazine methosulfate as an artificial electron acceptor . Further oxidation yielded 2-chloroacrylic acid . The organism also produced hydrolytic dehalogenases converting 2-chloroacetic acid and 2-chloropropionic acid.

J Cell Physiol, 1993 Feb, 154(2), 222 - 8
Involvement of the Golgi region in the intracellular trafficking of cholera toxin; Nambiar MP et al.; The intracellular pathway following receptor-mediated endocytosis of cholera toxin was studied using brefeldin A (BFA), which inhibited protein secretion and induced dramatic morphological changes in the Golgi region . In both mouse Y1 adrenal cells and CHO cells, BFA at 1 micrograms/ml caused a 80-90% inhibition of the cholera toxin (CT)-induced elevation of intracellular cAMP . The inhibition of the cytotoxicity of CT by BFA was also observed in a rounding assay of Y1 adrenal cells . The inhibition of CT cytotoxicity by BFA was dose dependent, with the ID50 value similar to the LD50 of BFA in Y1 adrenal cells . Binding and internalization of {125I}-labeled cholera toxin in Y1 adrenal cells was not affected by BFA . Unlike the BFA-sensitive cell lines such as Y1 adrenal and CHO cells, BFA at 1 micrograms/ml did not inhibit the cytotoxicity of CT in PtK1 cells, of which the Golgi structure was BFA-resistant . These results strongly suggest that a BFA-sensitive Golgi is required for the protection of CT cytotoxicity by BFA . In contrast, elevation of the intracellular cAMP by forskolin, which acts directly on the plasma membrane adenylate cyclase, was not affected by BFA . These observations indicate that the intoxication of target cells by CT requires an intact Golgi region for its intracellular trafficking and/or processing . In this respect, CT shares a common intracellular pathway with ricin, Pseudomonas toxin, and modeccin, even though their structures and modes of action are very different.

J Steroid Biochem Mol Biol, 1993 Feb, 44(2), 133 - 9
Cloning, DNA sequencing and expression of (3-17)beta hydroxysteroid dehydrogenase from Pseudomonas testosteroni; Abalain JH et al.; We describe the cloning, sequencing and overexpression of the (3-17)beta hydroxysteroid dehydrogenase gene of Pseudomonas testosteroni . A genomic library of Ps . testosteroni total DNA constructed from SauIIIA digests ligated to a lambda gt11 vector was probed with polyclonal antibody raised against purified enzyme . Subclones derived from a recombinant phage containing a 2661-base-pair insert were sequenced and found to contain an open reading frame of 765 base pairs that corresponds to a protein of 254 amino acid residues . A 1492-base-pair fragment was inserted into pBR322 plasmid vector and used to construct a strain of E . coli HB101 that overexpressed the steroid dehydrogenase gene.

J Thorac Cardiovasc Surg, 1993 Feb, 105(2), 247 - 52
An evaluation of the role of omentopexy and of early perioperative corticosteroid administration in clinical lung transplantation . The University of Toronto and Washington University Lung Transplant Programs; Miller JD et al.; Early success in clinical lung transplantation was believed due in part to the technique of bronchial anastomosis, routine bronchial omentopexy, and avoidance of early postoperative corticosteroid therapy . A recent 16-month consecutive experience at the University of Toronto and Washington University with single or bilateral lung transplantation was compared to study the current short-term effect of these perioperative strategies . At the University of Toronto, of 37 patients undergoing lung transplantation, 30 (group I) had telescoped bronchial anastomoses, coverage of the bronchus with local tissue only (no omentopexy), and routine perioperative corticosteroid administration . At Washington University, of 50 patients having lung transplantation, 44 (group II) had end-to-end bronchial anastomoses wrapped in omentum and received no routine perioperative corticosteroid . In group I, septic lung disease was the most frequent indication (14 of 29 patients), whereas in group II obstructive lung disease was the most frequently encountered condition (24 of 44 patients) . Sepsis accounted for three of five early deaths in group I (all due to resistant Pseudomonas cepacia infection in recipients who had cystic fibrosis) and for two of four perioperative deaths in group II (one Pseudomonas, and Candida) . In group I, cytomegalovirus prophylaxis was administered to all patients except recipients negative for cytomegalovirus receiving grafts from donors also negative for cytomegalovirus . Cytomegalovirus infection requiring treatment was encountered in 5 of 30 patients in group I in comparison with 23 of 44 recipients in group II where only D+ and R- mismatches received prophylaxis . Routine omentopexy is not essential for successful lung transplantation . Early postoperative corticosteroids do not impair airway healing, but neither do these agents appear to protect against acute rejection episodes . While routine corticosteroids do not predispose the recipient to cytomegalovirus infection, their use may increase the likelihood of postoperative bacterial sepsis.

Comp Biochem Physiol C, 1993 Feb, 104(2), 335 - 44
The metabolism of di(2-ethylhexyl)phthalate in the earthworm Lumbricus terrestris; Albro PW et al.; 1 . Earthworms can hydrolyze di-(2-ethylhexyl) phthalate (DEHP) to mono-2-ethylhexyl phthalate (MEHP) and phthalic acid (PA) . 2 . They apparently cannot produce the side-chain-oxidized derivatives of MEHP that constitute the major DEHP metabolites in higher animals . 3 . With the assistance of intestinal bacterial Pseudomonas, the worm-derived PA is degraded through protocatechuic and beta-carboxymuconic acids to CO2 . 4 . There is an indication of a second pathway for degradation of PA leading through benzoic acid.

Appl Environ Microbiol, 1993 Feb, 59(2), 458 - 66
DNA sequence and transcriptional analysis of the tblA gene required for tabtoxin biosynthesis by Pseudomonas syringae; Barta TM et al.; The tblA gene of Pseudomonas syringae is required for tabtoxin biosynthesis and is under the control of a regulatory gene, lemA . We have determined the nucleotide sequence of the tblA gene and identified the 5' end of the tblA gene transcript . The sequence of the tblA gene was identified to that of the recently reported open reading frame 1 gene of the tabA region of the BR2 chromosome . The open reading frame of the tblA gene potentially encodes a protein of 231 amino acids . mRNA from the tblA gene was detected at all phases of cells grown in minimal medium . This result is correlated with the constitutive production of tabtoxinine-beta-lactam (the biologically active part of the toxin) by P . syringae BR2R in minimal medium, as quantitated by a phenylisothiocyanate derivatization method.

Infect Immun, 1993 Feb, 61(2), 656 - 62
Ability of Pseudomonas pseudomallei malleobactin to acquire transferrin-bound, lactoferrin-bound, and cell-derived iron; Yang H et al.; The ability of malleobactin to mobilize iron from transferrin and lactoferrin was examined in an equilibrium dialysis assay in the absence of bacteria . Malleobactin was capable of removing iron from both transferrin and lactoferrin at pH values of 7.4, 6.0, and 5.0 . However, the levels of iron mobilization were greater for transferrin than for lactoferrin at all the pH values used in the assay . The ability of Pseudomonas pseudomallei to acquire iron from 30% iron-saturated transferrin and K562 human erythroleukemic cells was compared in parallel cultures as described previously (J . H . Brock, P . H . Williams, J . Liceaga, and K . G . Woldridge, Infect . Immun . 59:3185-3190, 1991) . P . pseudomallei U7 tended to acquire iron from transferrin . In contrast, P . aeruginosa PAO and P . cepacia Pc275C acquired iron from both sources . P . cepacia H1721, which does not produce detectable siderophores, but can utilize malleobactin, pyochelin, and azurechelin as iron sources, was used in a similar experiment . Addition of malleobactin resulted in iron uptake only from transferrin, whereas pyochelin and azurechelin promoted iron uptake from both sources . When the siderophores were incubated with K562 cells alone, malleobactin was less efficient at removing iron from cells than pyochelin and azurechelin . It was also determined that malleobactin was less effective in binding to or entering cells than pyochelin and azurechelin . These results suggest that malleobactin can acquire iron more effectively from host proteins than from cellular sources . Pyochelin and azurechelin can acquire cell-derived iron in addition to iron bound to host proteins.

Plant Physiol, 1993 Feb, 101(2), 441 - 50
An Arabidopsis thaliana lipoxygenase gene can be induced by pathogens, abscisic acid, and methyl jasmonate; Melan MA et al.; We isolated and characterized a 2.8-kb, full-length, Arabidopsis thaliana cDNA clone encoding a lipoxygenase . DNA sequence analysis showed that the deduced amino acid sequence of the Arabidopsis protein is 72 to 78% similar to that of legume seed lipoxygenases . DNA blot analysis indicated that Arabidopsis contains a single gene, LOX1, with appreciable homology to the cDNA clone . RNA blot analysis showed that the LOX1 gene is expressed in Arabidopsis leaves, roots, inflorescences, and young seedlings . LOX1 expression levels were highest in roots and young seedlings . In mature plants, LOX1 mRNA levels increased upon treatment with the stress-related hormones abscisic acid and methyl jasmonate and remained high for at least 96 h . Expression of the LOX1 gene was examined following infiltration of leaves with virulent (Psm ES4326) and avirulent (Pst MM1065) strains of Pseudomonas syringae . LOX1 mRNA levels were induced approximately 6-fold by both virulent and avirulent strains; however, the response to avirulent strains was much more rapid . Infiltration of leaves with Pst MM1065 resulted in maximal induction within 12 h, whereas maximal induction by Psm ES4326 did not occur until 48 h . When a cloned avr gene, avrRpt2, was transferred to Psm ES4326, LOX1 mRNA accumulated in a pattern similar to that observed for the avirulent strain Pst MM1065.

Biochem Biophys Res Commun, 1993 Jan 29, 190(2), 509 - 15
Improved purification of steroid 1:2-dehydrogenase from Nocardia opaca and partial characterization of its cloned gene sequence; Drobnic K et al.; We have purified a steroid-inducible 1:2-dehydrogenase from Nocardia opaca . The final enzyme preparation was purified 120-fold with a recovery of 38% . The N-terminal amino acid sequence was determined to be: Met-Gln-Asp-Trp-Thr-Ser-Glu-(Cys)-Asp-Val-Leu-Val-Val-Gly- . From the genomic library of Nocardia opaca in the plasmid pUC19, a clone designated as pSTD23 containing a 0.9 kb KpnI-PstI fragment was found to hybridize with an oligonucleotide probe corresponding to the first six amino acids from the N-terminal of the purified protein . The nucleotide sequence of the upstream region and a part of the structural domain were determined . The sequence of the first 56 amino acids of the steroid 1:2-dehydrogenase from Nocardia opaca as deduced from its gene sequence showed a 58% homology with the corresponding gene from Pseudomonas testosteroni, and the conservative sequences in the FAD-binding domain were also determined.

FEBS Lett, 1993 Jan 25, 316(2), 119 - 22
Lipid transfer proteins (nsLTPs) from barley and maize leaves are potent inhibitors of bacterial and fungal plant pathogens; Molina A et al.; Four homogeneous proteins (Cw18, Cw20, Cw21, Cw22) were isolated from etiolated barley leaves by extraction of the insoluble pellet from a Tris-HCl (pH 7.5) homogenate with 1.5 M LiCl and fractionation by reverse-phase high-performance liquid chromatography . All 4 proteins inhibited growth of the pathogen Clavibacter michiganensis subsp . sepedonicus (EC50s = 1-3 x 10(-7) M) and had closely related N-terminal amino acid sequences . The complete amino acid sequences of proteins Cw18 and Cw21 were determined and found to be homologous to previously described, non-specific lipid transfer proteins from plants (32-62% identical positions) . The proteins also inhibited growth of the bacterial pathogen Pseudomonas solanacearum (EC50s = 3-6 x 10(-7) M) and the fungus Fusarium solani (EC50s = 3-20 x 10(-6) M) . A homologous protein from maize leaves (Cw41) was purified in a similar manner and also found to have inhibitory properties . A synergistic effect against the fungus was observed when protein Cw21 was combined with thionins . A defense role for non-specific lipid transfer proteins from plants is proposed.

Proc Natl Acad Sci U S A, 1993 Jan 15, 90(2), 547 - 51
A recombinant immunotoxin that is active on prostate cancer cells and that is composed of the Fv region of monoclonal antibody PR1 and a truncated form of Pseudomonas exotoxin; Brinkmann U et al.; Monoclonal antibody PR1 binds to the surface of normal prostate cells and to adenocarcinomas of the prostate . The cDNAs coding for the heavy and light chain variable regions of monoclonal antibody PR1 were cloned by PCR techniques . A recombinant toxin was then constructed that has the heavy chain variable region of monoclonal antibody PR1 connected to the light chain variable region by a flexible peptide linker to create a single-chain Fv; the Fv in turn is fused to a truncated form of Pseudomonas exotoxin . The resulting recombinant immunotoxin PR1(Fv)-PE38KDEL was produced in Escherichia coli and accumulated in inclusion bodies . After denaturation and renaturation, active monomeric molecules with a molecular mass of approximately 65 kDa were purified to homogeneity . PR1(Fv)-PE38KDEL binds specifically to cells containing the PR1 antigen and is very cytotoxic toward a subset of LNCaP cells that express the PR1 antigen on their surface.

Cancer Res, 1993 Jan 15, 53(2), 340 - 7
Immunotoxins made with a recombinant form of Pseudomonas exotoxin A that do not require proteolysis for activity; Theuer CP et al.; We used recombinant DNA technology to construct a mutant form of Pseudomonas exotoxin A (PE) called cysPE35 that contains amino acids 280-364 and 381-613 of PE . cysPE35 begins at the native PE proteolytic cleavage site and contains a single cysteine residue at position 287 that can be used to conjugate the toxin to monoclonal antibodies (MAbs) . Unlike immunotoxins containing larger mutant forms of PE, such as PE40 or PE38, immunotoxins containing cysPE35 linked through a disulfide bond do not require proteolysis to generate a toxin fragment able to translocate to the cytosol . cysPE35 was conjugated to several MAbs and their activities were studied in vitro and in vivo . The concentration of toxin that inhibited protein synthesis as measured by a decrease in {3H}leucine incorporation of 50% of cysPE35 conjugated through a disulfide bond to the MAb HB21, which targets the human transferrin receptor, was 1 ng/ml on A431 cells . The MAb HB21 conjugated through a thioether bond to cysPE35 was much less active (concentration of toxin that inhibited protein synthesis as measured by a decrease in {3H}leucine incorporation of 50%, 200 ng/ml) . An immunotoxin containing PE38 conjugated through either a disulfide or thioether bond to the MAb HB21 had a concentration of toxin that inhibited protein synthesis as measured by a decrease in {3H}leucine incorporation of 50% of 5 ng/ml, indicating that proteolysis of PE38 may be rate limiting in the action of these immunotoxins . Two other MAbs, LL2 and B3, were also conjugated through a disulfide bond to cysPE35 . Both immunotoxins were also more active against cultured cells than conjugates using PE38 or PE40, and caused complete regression of human tumor xenografts growing in nude mice . In conclusion, we have constructed a mutant form of PE which must be coupled to MAbs through a disulfide bond to produce fully active immunotoxins that do not require proteolysis to generate a toxin fragment able to reach the cell cytosol.

Cancer Res, 1993 Jan 15, 53(2), 334 - 9
BR96 sFv-PE40, a potent single-chain immunotoxin that selectively kills carcinoma cells; Friedman PN et al.; We have constructed a single-chain immunotoxin composed of the carcinoma-reactive antibody BR96 and a truncated form of Pseudomonas exotoxin . The chimeric molecule, BR96 sFv-PE40, was expressed in Escherichia coli and localized to the inclusion bodies . We purified and identified two species of BR96 sFv-PE40, monomers and aggregates . The monomeric form was able to bind well to the BR96 antigen, a Lewisy-related antigen, while the aggregate was not . The binding affinity of the monomeric recombinant immunotoxin was 5-fold less than intact BR96 IgG, and its specificity for the BR96 antigen was confirmed by competition analysis . Monomeric BR96 sFv-PE40 was found to be extremely cytotoxic against cancer cells displaying the BR96 antigen . The cytotoxicity of the fusion protein correlates directly with antigen density on the tumor cell lines tested . The breast carcinoma cell line MCF-7, which has the highest density of BR96 antigen, was the most sensitive to BR96 sFv-PE40, with a concentration producing 50% protein synthesis inhibition of 5 pM . BR96 sFv-PE40 was found to have a t1/2 in serum of 28.5 min in athymic mice, compared to that of the chemical conjugate, chiBR96-LysPE40, which was 54 min . These data indicate that the single-chain immunotoxin BR96 sFv-PE40 is a potent inhibitor of protein synthesis in target cell lines and may be an effective agent for the treatment of cancer.

Gene, 1993 Jan 15, 123(1), 17 - 24
Analysis of Pseudomonas gene products using lacIq/Ptrp-lac plasmids and transposons that confer conditional phenotypes; de Lorenzo V et al.; Novel transposon and plasmid-based broad-host-range expression systems have been developed to facilitate the genetic analysis of gene products of Pseudomonas and related Gram- bacteria . The properties of lacIq/Ptrp-lac were used to construct mini-Tn5 expression vector transposons and RSF1010-derived plasmids for controlled expression and generation of conditional phenotypes . These plasmids were used to hyper-express the XylS regulator of the meta operon of the TOL plasmid of P . putida or the bphB and bphC genes of the polychlorobiphenyl-degrading pathway of Pseudomonas sp . LB400 in different strains of Pseudomonas instead of in Escherichia coli . Specific activity of 2.3 dihydroxybiphenyl dioxygenase (bphC gene product) was increased tenfold when hyperproduced in its native host as compared to E . coli, but under the same in vivo conditions, the XylS regulator formed protein aggregates . The other lacIq/Ptrp-lac-based expression vector presented here, transposon mini-Tn5 lacIq/Ptrc, facilitates the insertion of genetic cassettes containing heterologous genes under the control of lac inducers in the chromosome of target bacteria, as shown by monitoring expression of a lacZ reporter cloned in mini-Tn5 lacIq/Ptrc and inserted in the chromosome of P . putida.

Virology, 1993 Jan, 192(1), 375 - 9
Primary HIV-1 isolates refractory to neutralization by soluble CD4 are potently inhibited by CD4-Pseudomonas exotoxin; Kennedy PE et al.; Despite the ability of soluble forms of CD4 (sCD4) and related CD4 derivatives to neutralize human immunodeficiency type 1 (HIV-1) infectivity in vitro, these agents have shown little evidence of efficacy in clinical trials with infected individuals . These disappointing findings may be related to recent observations that much higher concentrations of sCD4 are required for in vitro neutralization of primary HIV-1 isolates compared to laboratory-adapted strains . An alternative CD4-based therapeutic strategy exploits CD4 as a targeting agent to direct cytotoxic molecules to selectively kill HIV-infected cells . In this report we demonstrate that CD4-Pseudomonas exotoxin inhibits spreading infection by primary HIV-1 isolates known to be highly refractory to neutralization by soluble CD4; the observed potency is at least as great as for a prototypic sCD4-sensitive, laboratory-adapted HIV-1 strain . Thus, the in vitro efficacy of a CD4-based agent, which acts by targeted killing of infected cells, appears not to be compromised by features which render primary HIV-1 isolates refractory to neutralization by sCD4 derivatives . These results have important conceptual and practical implications for CD4-based therapeutic strategies.

Cancer Invest, 1993, 11(3), 252 - 7
Depletion of patients' plasma tryptophan using tryptophan side-chain oxidase columns; Ho DH et al.; The use of the enzyme tryptophan side-chain oxidase, isolated from Pseudomonas XA, was explored in 3 patients with refractory acute lymphocytic leukemia . Patients were given either a low-tryptophan diet or tryptophan-free hyperalimentation, prior to and during therapy . Their plasma, separated by pheresis, was continuously passed through a tryptophan depletion column containing the immobilized tryptophan side-chain oxidase . Up to 4 plasma volumes were passed through the column daily, 5 days per week for 2-3 weeks, and plasma tryptophan levels, both free and total, were measured by high-performance liquid chromatography . Pre- and postcolumn plasma samples were collected throughout the pheresis procedure . All postcolumn plasma samples had unmeasurable tryptophan levels throughout the treatment period, whereas precolumn samples were always measurable . Generally, tryptophan levels of plasma isolated from peripheral blood decreased after therapy, but rebounded by the next day . The enzyme depletion column reduces circulating plasma tryptophan levels, and its use is well tolerated by patients . However, further development of this method will require study of the effects of diet and of the duration, interval, and frequency of use of this column on therapeutic efficacy . Problems include difficulties with extended diet compliance and apparently intensive mobilization of tryptophan from body stores, which may preclude the clinical application of this enzyme depletion column.

Antonie Van Leeuwenhoek, 1993 Jan, 63(1), 55 - 62
Regulation of the expression of the Pseudomonas stutzeri recA gene; Vosman B et al.; With the aid of recA-lacZ fusion strains, the in vivo regulation of the Pseudomonas stutzeri recA gene has been studied . It is shown that expression of this gene can be induced with a variety of DNA damaging agents, as well as with agents that interfere with DNA replication . For this induction, the presence of an active RecA protein is essential . Sequence analysis of the promoter region of the P . stutzeri recA gene showed that its open reading frame is preceded by an SOS-box, suggesting a regulation of its expression, similar to the regulation of recA expression in Escherichia coli.

An Med Interna, 1993 Jan, 10(1), 27 - 30
{Hematogenous pubic osteoarthritis}; Cabo Cabo J et al.; Pubic osteoarthritis is a little known pathological entity with a very controversial etiology . We present four cases of public osteoarthritis in which the infectious etiology was hematogeneous . The causal germs were: Staphilococcus aureus, Pseudomona aeruginosa, Mycobacterium tuberculosis and Brucella melitensis . In the first three cases, a surgical approach was used, allowing us to establish the etiological diagnosis of the process and to perform the local debridement . In the case of brucellar etiology, an isolated medical treatment was applied, according to the therapeutical guidelines recommended for brucellar bone infections . We have not observed recurrence of the septic process in any of the four cases, with a follow-up period ranging from one to three years.

Klin Padiatr, 1993 Jan-Feb, 205(1), 18 - 22
{Aminoglycosides in patients with mucoviscidosis and pulmonary exacerbation . Comparison of once or three times daily administration}; Heininger U et al.; Twenty-six patients with cystic fibrosis and pulmonary exacerbations were enrolled in a prospective randomized study to compare the efficacy of aminoglycosides (tobramycin or netilmicin) administered once daily (21 episodes, 5 with netilmicin, 16 with tobramycin) and thrice daily (23 episodes, 2 with netilmicin, 21 with tobramycin), respectively . In addition, the patients received an anti-pseudomonal beta-lactam antibiotic . In the single-dose group the total daily dosage was 4.97 +/- 1.12 mg/kg (total dosage per exacerbation: 74.55 mg/kg), compared to 9.60 +/- 2.70 mg/kg in the triple-dose group (total dosage per exacerbation: 165.12 mg/kg) . The mean peak and trough serum levels of the aminoglycoside were 8.31 +/- 1.76 mg/l and 0.18 +/- 0.10 mg/l, respectively in the single dose group compared to 6.12 +/- 1.30 mg/l and 0.58 +/- 0.31 mg/l in the triple dose group . Success of treatment, defined as decrease in leucocyte counts, normalization of elevated CRP-values, number of days in hospital and interval until next admission to hospital, was not different between both groups . We conclude that single daily dose of aminoglycosides was as efficacious as triple dose in our patients.

J Antibiot (Tokyo), 1993 Jan, 46(1), 135 - 40
Biphenomycin C, a precursor of biphenomycin A in mixed culture; Ezaki M et al.; A precursor of biphenomycin A in mixed culture of Streptomyces griseorubiginosus No . 43708 with Pseudomonas maltophilia No . 1928 was isolated and characterized . The structure of the precursor, designated biphenomycin C was determined to be a peptide which is composed of biphenomycin A and arginylserine residue (Fig . 1), on the basis of chemical and spectroscopic evidence.

Antimicrob Agents Chemother, 1993 Jan, 37(1), 123 - 5
In vitro activities of meropenem, PD 127391, PD 131628, ceftazidime, chloramphenicol, co-trimoxazole, and ciprofloxacin against Pseudomonas cepacia; Lewin C et al.; In a study of 110 Pseudomonas cepacia isolates from patients without cystic fibrosis, the in vitro potencies of three new compounds, meropenem, PD 127391, and PD 131628, were comparable to those of ceftazidime and ciprofloxacin and exceeded those of chloramphenicol and co-trimoxazole . The MICs of ceftazidime, ciprofloxacin, meropenem, and the PD compounds for 90% of strains tested were < or = 4 micrograms/ml, whereas they were 32 micrograms/ml for chloramphenicol and co-trimoxazole . Data for 20 isolates from patients with cystic fibrosis indicated that the isolates were less susceptible to all seven antibiotics tested, with the most active compounds being meropenem and PD 127391.

J Steroid Biochem Mol Biol, 1993 Jan, 44(1), 101 - 4
Novel oxidative cleavage of C17-C20 bond in pregnane by a Pseudomonas sp; Dhar A et al.; Oxidative cleavage of the C17-C20 bond in progesterone by Pseudomonas sp . is reported . Transformation occurred under in vivo conditions . The bioconverted products were characterized as 1,4-androstadien-3,17-dione and 17 beta-hydroxy-1,4-androstadien-3-one . The steroid nucleus was not further degraded although the test organism had the capacity to induce dehydrogenation at C1 of this alpha, beta-conjugated steroid.

J Clin Invest, 1993 Jan, 91(1), 88 - 93
Expression of high affinity interleukin-4 receptors on human renal cell carcinoma cells and inhibition of tumor cell growth in vitro by interleukin-4; Obiri NI et al.; Previously, Puri et al . (Puri, R . K., M . Ogata, P . Leland, G . M . Feldman, D . Fitzgerald, and I . Pastan . 1991 . Cancer Res . 51:3011-3017) have demonstrated that murine sarcoma and colon adenocarcinoma cells express high affinity interleukin-4 receptors (IL-4R) which are internalized after binding to a chimeric ligand consisting of IL-4 and Pseudomonas exotoxin . In the present study, we have tested primary cultures of human renal cell carcinoma (RCC) cells, generated from tumor specimens obtained after nephrectomy, for the expression of IL-4R and their modulation by IL-4 . By using iodinated IL-4 in a receptor binding assay, we observed that renal cell carcinoma cells expressed a single class of high affinity IL-4R ranging from 1,425 +/- 207 (mean +/- SEM) to 3,831 +/- 299 (mean +/- SEM) IL-4R molecules/cell with a Kd ranging from 112 +/- 11 pM to 283 +/- 71 pM . Northern blot analysis for IL-4R gene expression, performed with a cDNA probe to IL-4R, revealed that all RCC cells exhibited a single mRNA species of 4 kb . IL-4 downregulated the surface expression of IL-4R on one RCC tumor cell line . The function of IL-4R expression on RCC tumor cells was further determined by investigating the effect of IL-4 on tumor cell growth in vitro and comparing it with IL-4 effect on growth of normal fibroblast and endothelial cell lines . Tumor cell growth, as measured by {3H}thymidine incorporation, was inhibited by IL-4 from 20 to 68% in a dose-dependent manner . A neutralizing antibody to human IL-4 was able to reverse the growth inhibitory effect of IL-4 . Normal human fibroblast and endothelial cell lines also expressed high affinity IL-4R, however, IL-4 did not inhibit their growth in vitro . In fact, IL-4 caused modest stimulation of their growth . Taken together, our findings can help develop strategies for the treatment of RCC in which IL-4R may be used as a target for IL-4 itself, for IL-4 toxin therapy or, alternatively, in gene therapy.

J Bacteriol, 1993 Jan, 175(2), 395 - 400
Oxidation of biphenyl by a multicomponent enzyme system from Pseudomonas sp . strain LB400; Haddock JD et al.; Pseudomonas sp . strain LB400 grows on biphenyl as the sole carbon and energy source . This organism also cooxidizes several chlorinated biphenyl congeners . Biphenyl dioxygenase activity in cell extract required addition of NAD(P)H as an electron donor for the conversion of biphenyl to cis-2,3-dihydroxy-2,3-dihydrobiphenyl . Incorporation of both atoms of molecular oxygen into the substrate was shown with 18O2 . The nonlinear relationship between enzyme activity and protein concentration suggested that the enzyme is composed of multiple protein components . Ion-exchange chromatography of the cell extract gave three protein fractions that were required together to restore enzymatic activity . Similarities with other multicomponent aromatic hydrocarbon dioxygenases indicated that biphenyl dioxygenase may consist of a flavoprotein and iron-sulfur proteins that constitute a short electron transport chain involved in catalyzing the incorporation of both atoms of molecular oxygen into the aromatic ring.

J Bacteriol, 1993 Jan, 175(2), 377 - 85
Purification and properties of the physically associated meta-cleavage pathway enzymes 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating) from Pseudomonas sp . strain CF600; Powlowski J et al.; The final two steps in the dmp operon-encoded meta-cleavage pathway for phenol degradation in Pseudomonas sp . strain CF600 involve conversion of 4-hydroxy-2-ketovalerate to pyruvate and acetyl coenzyme A (acetyl-CoA) by the enzymes 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating) {acetaldehyde:NAD+ oxidoreductase (CoA acetylating), EC 1.2.1.10} . A procedure for purifying these two enzyme activities to homogeneity is reported here . The two activities were found to copurify through five different chromatography steps and ammonium sulfate fractionation, resulting in a preparation that contained approximately equal proportions of two polypeptides with molecular masses of 35 and 40 kDa . Amino-terminal sequencing revealed that the first six amino acids of each polypeptide were those deduced from the previously determined nucleotide sequences of the corresponding dmp operon-encoded genes . The isolated complex had a native molecular mass of 148 kDa, which is consistent with the presence of two of each polypeptide per complex . In addition to generating acetyl-CoA from acetaldehyde, CoA, and NAD+, the dehydrogenase was shown to acylate propionaldehyde, which would be generated by action of the meta-cleavage pathway enzymes on the substrates 3,4-dimethylcatechol and 4-methylcatechol . 4-Hydroxy-2-ketovalerate aldolase activity was stimulated by the addition of Mn2+ and, surprisingly, NADH to assay mixtures . The possible significance of the close physical association between these two polypeptides in ensuring efficient metabolism of the short-chain aldehyde generated by this pathway is discussed.

J Virol, 1993 Jan, 67(1), 593 - 5
A mutant CHO-K1 strain with resistance to Pseudomonas exotoxin A is unable to process the precursor fusion glycoprotein of Newcastle disease virus; Inocencio NM et al.; RPE.40, a mutant strain of CHO-K1 cells isolated for resistance to Pseudomonas exotoxin A and cross-resistant to alphaviruses, is also highly resistant to virulent strains of Newcastle disease virus . The resistance of RPE.40 cells to Newcastle disease virus results from the failure to cleave the viral envelope precursor glycoprotein Fo to fusion glycoprotein F1 at the consensus sequence (Lys/Arg)-Arg-Gln-(Lys/Arg)-Arg.

Cancer Detect Prev, 1993, 17(2), 289 - 93
Recombinant immunotoxins: new therapeutic agents for cancer treatment; Pastan IH; We have developed new agents for the treatment of cancer by genetically modifying Pseudomonas exotoxin . We have deleted the cell-binding region of Pseudomonas exotoxin and replaced it with various growth factors or the combining regions of antibodies in a single chain form . These new recombinant molecules are called recombinant toxins . Several different types of recombinant toxins have been produced . B3(Fv)-PE38KDEL is a recombinant toxin that kills many different adenocarcinomas and epidermoid carcinomas . The molecule is now undergoing preclinical development.

Perit Dial Int, 1993, 13 Suppl 2, S338 - 40
Peritoneal catheter loss and death in continuous ambulatory peritoneal dialysis peritonitis: correlation with clinical and biochemical parameters; Tzamaloukas AH et al.; Clinical and biochemical parameters associated with the removal of the peritoneal catheter and death following continuous ambulatory peritoneal dialysis (CAPD) peritonitis were analyzed in 120 episodes of peritonitis . Episodes resulting in catheter removal (n = 24, 20%) and those ending in patient death (n = 12, 10%) were respectively compared with episodes in which peritoneal catheters were saved and from which the patients survived . Variables associated with catheter removal included advanced age, long duration of peritonitis, coexisting exit-site/tunnel infection, infection caused by pseudomonas or fungi, elevated aspartate aminotransferase (AST) and malnutrition at presentation with peritonitis (serum albumin 29.5 +/- 7.6 g/L vs 33.8 +/- 4.8 g/L in episodes in which the catheters were saved, p = 0.014), and worsening malnutrition during peritonitis . Variables associated with death from peritonitis included diabetes mellitus, persistence of the infection, removal of the peritoneal catheter, infection with pseudomonas, malnutrition prior to the infection (serum albumin 29.5 +/- 3.2 g/L vs 34.7 +/- 4.2 g/L in survivors, p < 0.001), presentation with elevated AST and worsening malnutrition, and the development of pronounced malnutrition during infection (serum albumin 18.1 +/- 4.1 g/L vs 28.9 +/- 5.8 g/L in survivors, p < 0.001) . Deaths were caused primarily by cardiovascular events . Both removal of the peritoneal catheter and death as consequences of CAPD peritonitis are associated with malnutrition and pseudomonas infection . In addition, death is more frequent in diabetic patients.

Int Ophthalmol Clin, 1993 Winter, 33(1), 23 - 49
Contact lens-related infectious keratitis; Palmer ML et al.; Infectious keratitis is the most serious complication of contact lens use . Virtually all contact lens wearers are at risk . Initial therapy consists of frequent broad-spectrum fortified antibiotic drops after appropriate laboratory workup . Pseudomonas and Acanthamoeba species are the most important causes of contact lens-associated ulcers . Acanthamoeba keratitis produces significant ocular morbidity, and treatment is not always effective . Recent studies have provided new insights regarding the incidence, risk factors, and pathogenesis of contact lens-related infectious keratitis . Extended-wear soft contact lens wearers are at greatest risk . With our present understanding of the pathogenesis and risk factors of contact lens-related infectious keratitis, daily-wear schedules are strongly advised . Even under the best of lens care conditions, infectious keratitis may still occur . It is therefore imperative that patients be informed to remove their lenses and seek medical evaluation if any discomfort develops.

Folia Microbiol (Praha), 1993, 38(5), 376 - 8
Degradation of 2-chlorobenzoic and 2,5-dichlorobenzoic acids in soil columns by Pseudomonas stutzeri; Kozlovsky SA et al.; The heterocontinuous flow cultivation technique was used for the study of 2-chlorobenzoic and 2,5-dichlorobenzoic acid degradation in soil columns inoculated with Pseudomonas stutzeri . 2-Chlorobenzoic and 2,5-dichlorobenzoic acids disappeared from the soil columns within 8 and 12 d, respectively . The presence of the haloaromatics increased the survival of strain KS25 in soil . Viable cell numbers in the soil columns flushed with 2-chlorobenzoic and 2,5-dichlorobenzoic acids were 1.3 and 2 times higher, respectively, than those without the chlorobenzoic acids after 30 d of incubation.

J Basic Microbiol, 1993, 33(4), 219 - 25
Dodecylguanidine monoacetate (dodine) causes severe membrane damage in Pseudomonas syringae above the critical micelle concentration; Cabral JP; The release of K+ from Pseudomonas syringae cells treated with dodecylguanidine monoacetate (dodine) was followed with a K(+)-selective glass electrode . Treatment of the cells with 5-15 mumol/l dodine resulted in low levels of K+ release, but higher surfactant concentrations caused extensive and rapid K+ efflux . Dodine concentrations that caused high K+ release also induced significant leakage of inorganic phosphate . The addition of 5-10 mumol/l dodine also caused an increase in the rate of oxygen consumption in the presence of glycerol or succinate, but an increase in concentration from 10 to 40 mumol/l resulted in a concomitant decrease in O2 consumption . The results from this and previous work suggest that dodine inhibits respiration firstly by causing drainage of coenzymes, and then by a direct interaction with the components of the respiratory chain . Previous work showed that above 25 mumol/l, dodine molecules aggregate to form micelles . The results therefore suggests that, in contrast with other cationic amphiphiles, the micellar form of dodine is more damaging to the cytoplasmic membrane than the free molecules.

Biosystems, 1993, 31(2-3), 169 - 80
Surface bacteria of Streblomastix strix are sensory symbionts; Dyer BD et al.; Streblomastix strix, a protist living in the hindguts of Zootermopsis sp . (termites) is covered with pseudomonad-like rod-shaped bacteria . These bacteria were demonstrated to be sensory (chemotactic) symbionts for S . strix . S . strix moved toward a source of sodium acetate, a presumed food molecule, when its bacteria were intact . If the bacteria were removed by antibiotic (carbenecillin) treatment, S . strix was unable to orient efficiently in a sodium acetate gradient . The mechanism of this interaction was not determined . This is the first case documented of a sensory symbiosis involving a surface layer of bacteria . However, it may not be an exceptional case given the large numbers of organisms with surfaces covered with symbionts . The assays for chemotaxis used in this research may be applied in other cases in which motile organisms are covered with symbionts.

Adv Perit Dial, 1993, 9, 206 - 10
Treatment and prevention of relapses of CAPD Pseudomonas peritonitis; Pasadakis P et al.; Pseudomonas peritonitis in continuous ambulatory peritoneal dialysis (CAPD) can be difficult to eradicate, because it is frequently resistant to common antibiotics, inducing the loss of the peritoneal cavity in some cases . A total of 14 episodes of Pseudomonas peritonitis in 12 patients (6 male, 6 female) were treated with intraperitoneal (IP) administration of a combination of ceftazidime and tobramycin . All patients were hospitalized . The loading doses were 1000 mg/2 L of ceftazidime and 1.7 mg/kg of tobramycin, and the maintenance IP doses were 250 mg/2 L of ceftazidime and 16 mg/2 L of tobramycin . The therapy duration was 14 days . In 7 episodes (group A) no other antibiotic regimen was provided, while in the remaining 7 episodes (group B) therapy was continued with 500 mg b.i.d . of oral ciprofloxacin for the next 14 days . Pseudomonas species isolated in group A were P . alcaligenis (1), P . putida (1), P . maltophilia (1), R . cepacia (1), and unidentified (3) . In group B the following Pseudomonas species were isolated: P . aeruginosa (4), P . diminuta (1), P . stutszeri (1), and unidentified (1) . Recurrence of peritonitis was seen in 4 episodes of group A with 2 catheter removals, while all episodes were cured in group B . These results suggest that IP ceftazidime and tobramycin with the additional use of oral ciprofloxacin is successful in the treatment and prevention of relapses of Pseudomonas peritonitis.

Cancer Treat Res, 1993, 68, 145 - 60
Recombinant fusion toxins--a new class of targeted biologic therapeutics; Woodworth TG et al.; The design and construction of a new class of recombinant therapeutic agents, receptor-specific cytotoxins, has occurred within the last 5 years . Development of a number of receptor-targeted fusion toxins has been based on a detailed understanding of the structure-function relationships of both diphtheria toxin and Pseudomonas exotoxin A, and availability of the nucleic acid sequences of each structural gene . A variety of fusion toxins in which the native receptor-binding domain of either diphtheria toxin or Pseudomonas exotoxin A has been genetically replaced with either a polypeptide hormone or growth factor have been constructed . These fusion toxins selectively intoxicate receptor-bearing cells in vitro and are active in a variety of animal model systems . DAB486IL-2, and IL-2 receptor targeted cytotoxin, is the first fusion toxin to be evaluated in patients . Phase I/II clinical trials have been performed in refractory leukemia/lymphoma, severe rheumatoid arthritis, and Type 1 diabetes . DAB486IL-2 has been administered to more than 200 patients, has been well tolerated, and has shown encouraging signs of potential efficacy in all three clinical indications . Thus, DAB486IL-2 represents a new class of targeted biological therapeutic response modifiers whose mode of action is based on selective elimination of target cells.

Microbiol Immunol, 1993, 37(7), 531 - 6
Priming effect of pseudomonal leukocidin on chemiluminescence response of rabbit polymorphonuclear leukocytes; Nishiya H et al.; To clarify effects of pseudomonal leukocidin (42.5 kd) on chemiluminescence (CL) production of polymorphonuclear leukocytes (PMNs), rabbit PMNs were stimulated by zymosan or phorbol myristate acetate (PMA) after pretreatment with the leukocidin, which by itself stimulated little chemiluminescence response . The extent of CL responses stimulated by zymosan or PMA was respectively 5.3- or 3.5-fold greater in leukocidin (1.5 micrograms/ml)-pretreated PMNs than in non-pretreated ones . The priming effect of the leukocidin was greater than that of G-CSF and related to some steps before NADPH oxidase activation . The increased CL productions might be related to tissue damages caused by pseudomonal infections in vivo.

Biometals, 1993 Summer, 6(2), 93 - 100
Ornibactins--a new family of siderophores from Pseudomonas; Stephan H et al.; Novel linear hydroxamate/hydroxycarboxylate siderophores from strains of Pseudomonas cepacia were isolated and named ornibactins . The ornibactins represent modified tetrapeptide siderophores, possessing the sequence L-Orn1(N delta-OH, N delta-acyl)-D-threo-Asp(beta-OH)-L-Ser-L-Orn4(N delta-OH, N delta-formyl)-1,4-diaminobutane . The N delta-acyl groups of Orn1(N delta-OH, N delta-acyl) may vary and represent the three acids 3-hydroxybutanoic acid, 3-hydroxyhexanoic acid and 3-hydroxyoctanoic acid, leading to a mixture of three different ornibactins, designated according to their acyl chain length as ornibactin-C4, ornibactin-C6 and ornibactin-C8 . Each of the siderophores is accompanied by a small amount of a more hydrophilic component with a 16 a.m.u . higher mass . The structure elucidation was based on results from gas chromatography amino acid analysis, electrospray mass spectrometry, and one- and two-dimensional nuclear magnetic resonance techniques.

Arch Microbiol, 1993, 159(4), 323 - 9
Degradation of diphenylether by Pseudomonas cepacia Et4: enzymatic release of phenol from 2,3-dihydroxydiphenylether; Pfeifer F et al.; 2,3-Dihydroxybiphenyl dioxygenase from Pseudomonas cepacia Et 4 was found to catalyze the ring fission of 2,3-dihydroxydiphenylether in the course of diphenylether degradation . The enzyme was purified and characterized . It had a molecular mass of 240 kDa and is dissociated by SDS into eight subunits of equal mass (31 kDa) . The purified enzyme was found to be most active with 2,3-dihydroxybiphenyl as substrate and showed moderate activity with 2,3-dihydroxydiphenylether, catechol and some 3-substituted catechols . The Km-value of 1 microM for 2,3-dihydroxydiphenylether indicated a high affinity of the enzyme towards this substrate . The cleavage of 2,3-dihydroxydiphenylether by 2,3-dihydroxybiphenyl dioxygenase lead to the formation of phenol and 2-pyrone-6-carboxylate as products of ring fission and ether cleavage without participation of free intermediates . Isotope labeling experiments carried out with 18O2 and H2(18)O indicated the incorporation of 18O from the atmosphere into the carboxyl residue as well as into the carbonyl oxygen of the lactone moiety of 2-pyrone-6-carboxylate . Based on these experimental findings the reaction mechanism for the formation of phenol and 2-pyrone-6-carboxylate is proposed in accordance with the mechanism suggested by Kersten et al . (1982).

Curr Microbiol, 1993 Jan, 26(1), 17 - 22
Antagonism of Pseudomonas cepacia against phytopathogenic fungi; Jayaswal RK et al.; Two strains of Pseudomonas cepacia, RJ3 and ATCC 52796, have been identified as potential antagonists of fungal plant pathogens . We have compared the antagonistic activity of these two strains against various fungal pathogens . Although both strains displayed high levels of antagonism, ATCC 52796 was slightly more antagonistic than RJ3 . The antagonist from RJ3 has been identified as the antifungal compound pyrrolnitrin after purification by HPLC and characterization by UV, IR, NMR, and mass spectroscopy . Both strains also antagonized the fungi by production of volatile compound(s), which have not yet been identified . Both strains are similar with respect to in vitro antagonism, mechanism of antagonism, and sensitivity to antibiotics.

J Infect Dis, 1993 Jan, 167(1), 230 - 3
Serology and carriage of Pseudomonas pseudomallei: a prospective study in 1000 hospitalized children in northeast Thailand; Kanaphun P et al.; Throat swab cultures and indirect hemagglutination assay (IHA) for Pseudomonas pseudomallei were done in 1000 randomly selected children at a large hospital in northeast Thailand . During 18 months, 17 children with melioidosis were admitted (0.46% of pediatric admissions excluding neonates born in the hospital) . Throat swab was positive for P . pseudomallei in 8 of these but in none of 1000 control children . IHA seroprevalence rose at a conversion rate of 24% per year, from 12% in those 1-6 months old to a plateau at approximately 80% after age 4 years . No control child < 4 had an IHA titer > 1:160 . The median titer in children with melioidosis was 1:80 (range, negative {in3}-1:5120) . Specificity of IHA declined with age, but high titers (> or = 1:640) remained diagnostically useful . Thus, throat carriage of P . pseudomallei indicates active melioidosis . There is no evidence for an asymptomatic carrier state in children . Environmental exposure to P . pseudomallei in endemic areas begins when the child becomes mobile.

J Cell Sci Suppl, 1993, 17, 229 - 33
Defective acidification of the biosynthetic pathway in cystic fibrosis; Barasch J et al.; Cystic fibrosis is associated with defective epithelial sodium chloride and fluid secretion in epithelia . In addition, there is widespread reductions in sialylation of secreted proteins and increases in the sulfation and fucosylation of mucus glycoproteins . The major morbidity in the disease is due to the colonization of respiratory epithelia by Pseudomonas . The cystic fibrosis gene (CFTR) is a cyclic AMP activated Cl channel, which when mutated is retained in the endoplasmic reticulum . We postulate that this Cl channel is responsible for effective acidification of the Golgi . In CF cells, we demonstrate the Golgi pH is higher than in normal cells and suggest that the abnormalities in glycoprotein biosynthesis is due to changes in the kinetics of sialyl transferase, a pH sensitive enzyme . Defects in sialylation also result in decreased sialylation of glycolipids and asialogangliosides are potential Pseudomonas receptors.

Acta Microbiol Pol, 1993, 42(2), 209 - 13
Attempts at the further characterisation of exoprotease from Pseudomonas sp . strain S9; Sklodowska A et al.; The correlation between EPS release and exoprotease activity from Pseudomonas sp . was shown . The presence of added exoprotease was recognized by cells of Pseudomonas sp . strains S9 and B29 and was a factor inhibiting exoprotease synthesis de novo . Examined enzyme is a metalloprotein containing zinc with mass estimated at 60,000 daltons.

Acta Microbiol Bulg, 1993, 30, 11 - 6
Virulence and susceptibility to phagocytosis of Pseudomonas pseudomallei R- and S-forms for ground squirrels (Citellus citellus L.); Velianov D et al.; Virulence and susceptibility to phagocytosis of Pseudomonas pseudomallei R- and S-forms for ground squirrels were studied . The alveolar macrophages and blood leucocytes (polymorphonuclear and mononuclear) was used . S-forms survived and multiplied intracellularly more actively in alveolar macrophages than R-forms and their virulence was considerably higher.

FEBS Lett, 1992 Dec 21, 314(3), 371 - 4
Brefeldin A inhibits protein synthesis in cultured cells; Fishman PH et al.; The fungal metabolite brefeldin A (BFA) is known to disrupt the Golgi apparatus resulting in redistribution of Golgi proteins to the endoplasmic reticulum and inhibition of protein secretion . BFA was found to inhibit protein synthesis in rat glioma C6 cells by up to 70% between 0.1 and 1 microgram/ml . Inhibition was both time-dependent and reversible . BFA inhibited protein synthesis to varying degrees in a number of other cell lines but not in BFA-resistant marsupial kidney cells . The same concentrations of BFA which inhibited protein synthesis, also blocked the inhibitory effects of Pseudomonas exotoxin and ricin on BFA-sensitive cells . BFA, however, was unable to block the inhibition of protein synthesis by the toxins in the resistant marsupial kidney cells.

N Engl J Med, 1992 Dec 17, 327(25), 1785 - 8
The prognostic value of exercise testing in patients with cystic fibrosis; Nixon PA et al.; BACKGROUND . Previous studies have shown female sex, impaired pulmonary function, older age, malnutrition, and colonization of the respiratory tract with Pseudomonas cepacia to be associated with a poor prognosis in patients with cystic fibrosis . We sought to determine the prognostic value of exercise testing in addition to the other prognostic factors . METHODS . A total of 109 patients with cystic fibrosis, 7 to 35 years old, underwent pulmonary-function and exercise testing in the late 1970s . They were followed for eight years to determine the factors associated with subsequent mortality . Survival rates were calculated with standard life-table methods . Cox proportional-hazards regression models were used to determine crude relative risks of mortality and relative risks adjusted for age, sex, body-mass index, forced expiratory volume in one second (FEV1) end-tidal partial pressure of carbon dioxide (PCO2) at peak exercise, and oxygen consumption at peak exercise (VO2 peak) . RESULTS . Patients with the highest levels of aerobic fitness (VO2 peak, > or = 82 percent of predicted) had a survival rate of 83 percent at eight years, as compared with rates of 51 percent and 28 percent for patients with middle (VO2 peak, 59 to 81 percent of predicted) and lowest (VO2 peak, < or = 58 percent of predicted) levels of fitness, respectively . After adjustment for other risk factors, patients with higher levels of aerobic fitness were more than three times as likely to survive than patients with lower levels of fitness . Colonization with P . cepacia was associated with a risk of dying that was increased fivefold . Age, sex, body-mass index, FEV1, and end-tidal PCO2 at peak exercise were not independently correlated with mortality . CONCLUSIONS . Higher levels of aerobic fitness in patients with cystic fibrosis are associated with a significantly lower risk of dying . Although better aerobic fitness may simply be a marker for less severe illness, measurement of VO2 peak appears to be valuable for predicting prognosis . Further research is warranted to determine whether improving aerobic fitness through exercise programs will result in a better prognosis.

FEMS Microbiol Lett, 1992 Dec 15, 79(1-3), 101 - 5
The new approaches to whole genome analysis of bacteria; Holloway BW et al.; A range of recombinant DNA techniques now enables whole genome analysis of any bacterium to be carried out without recourse to the classical means of bacterial genetic exchange . Using enzymes which cut infrequently, such as SpeI, combined with pulsed field gel electrophoresis, a physical map of ordered fragments can be constructed . By means of cloned fragments of known genes or oligonucleotides synthesized using data from DNA or protein sequence banks, the location of individual genes on this map can be determined . We have used these techniques to study whole genome structure in three species of Pseudomonas: P . aeruginosa, P . putida and P . solanacearum.

J Biol Chem, 1992 Dec 15, 267(35), 25396 - 401
Cell-mediated cleavage of Pseudomonas exotoxin between Arg279 and Gly280 generates the enzymatically active fragment which translocates to the cytosol; Ogata M et al.; Pseudomonas exotoxin (PE) is a three-domain toxin which is cleaved by a cellular protease within cells and then reduced to generate two prominent fragments (Ogata, M., Chaudhary, V . K., Pastan, I., and FitzGerald, D . J . (1990) J . Biol . Chem . 265, 20678-20685) . The N-terminal fragment is 28 kDa in size and contains the binding domain . The 37-kDa C-terminal fragment, which translocates to the cytosol, contains the translocation domain and the ADP-ribosylation domain . Cleavage followed by reduction is essential for toxicity since mutant forms of the toxin that cannot be cleaved by cells are nontoxic . Previous results with these mutants suggest that cleavage occurred in an arginine-rich (arginine residues are at positions 274, 276, and 279) disulfide loop near the beginning of the translocation domain, but the exact site of cleavage was not determined . Since very few molecules of the 37-kDa fragment are generated within cells it was not possible to determine the site of cleavage by performing a conventional N-terminal sequence analysis of the 37-kDa fragment . Two experimental approaches were used to overcome this limitation . First, existing amino acids near the cleavage sites were replaced with methionine residues; this was followed by the addition of {35S}methionine-labeled versions of these toxins to cells . The pattern of radioactive toxin fragments recovered from the cells indicated that the toxin was cleaved either just before or just after Arg279 . Second, {3H}leucine-labeled toxin was produced and added to the cells . Sequential Edman degradations were performed on the small amount of radioactive 37-kDa fragment that could be recovered from toxin-treated cells . A peak of radioactivity in the fifth fraction indicated that leucine was the 5th amino acid on the C-terminal side of the cleavage site . This result confirmed that cleavage was between Arg279 and Gly280.

Science, 1992 Dec 4, 258(5088), 1604 - 10
Phthalate dioxygenase reductase: a modular structure for electron transfer from pyridine nucleotides to {2Fe-2S}; Correll CC et al.; Phthalate dioxygenase reductase (PDR) is a prototypical iron-sulfur flavoprotein (36 kilodaltons) that utilizes flavin mononucleotide (FMN) to mediate electron transfer from the two-electron donor, reduced nicotinamide adenine nucleotide (NADH), to the one-electron acceptor, {2Fe-2S} . The crystal structure of oxidized PDR from Pseudomonas cepacia has been analyzed at 2.0 angstrom resolution resolution; reduced PDR and pyridine nucleotide complexes have been analyzed at 2.7 angstrom resolution . NADH, FMN, and the {2Fe-2S} cluster, bound to distinct domains, are brought together near a central cleft in the molecule, with only 4.9 angstroms separating the flavin 8-methyl and a cysteine sulfur ligated to iron . The domains that bind FMN and {2Fe-2S} are packed so that the flavin ring and the plane of the {2Fe-2S} core are approximately perpendicular . The {2Fe-2S} group is bound by four cysteines in a site resembling that in plant ferredoxins, but its redox potential (-174 millivolts at pH 7.0) is much higher than the potentials of plant ferredoxins . Structural and sequence similarities assign PDR to a distinct family of flavoprotein reductases, all related to ferredoxin NADP(+)-reductase.

FEMS Microbiol Lett, 1992 Dec 1, 78(2-3), 305 - 10
Pyrimidine base and ribonucleoside catabolic enzyme activities of the Pseudomonas diminuta group; West TP; Pyrimidine base and ribonucleoside catabolic enzyme activities of the two type strains of the Pseudomonas diminuta group were investigated for taxonomic classification purposes . The presence of the pyrimidine salvage enzyme nucleoside hydrolase was indicated in both type strains following thin-layer chromatographic analysis . The presence of the hydrolase was also confirmed by enzyme assay . In addition, the activities of the pyrimidine salvage enzymes dihydropyrimidine dehydrogenase and dihydropyrimidinase were measurable in cell-free extracts of both P . diminuta and P . vesicularis . An absence of cytosine deaminase activity was found when assaying extracts of the two type strains . Nucleoside hydrolase and dihydropyrimidine dehydrogenase levels in P . vesicularis were influenced by carbon source while dihydropyrimidinase activity was observed to increase after P . diminuta growth on dihydrothymine as a nitrogen source.

Microbiol Rev, 1992 Dec, 56(4), 662 - 76
Traits of fluorescent Pseudomonas spp . involved in suppression of plant root pathogens; O'Sullivan DJ et al.; Certain members of the fluorescent pseudomonad group have been shown to be potential agents for the biocontrol of plant root diseases . The major problems with the commercialization of these beneficial strains are that few wild-type strains contain all the desired characteristics for this process and the performance of strains in different soil and climatic conditions is not reproducible . Consequently, prior to selection and/or improvement of suitable strains for biocontrol purposes, it is necessary to understand the important traits required for this purpose . The production of fluorescent siderophores (iron-binding compounds) and antibiotic compounds has been recognized as important for the inhibition of plant root pathogens . Efficient root colonization is also a prerequisite for successful biocontrol strains . This review discusses some of the characteristics of fluorescent pseudomonads that have been suggested to be important for biocontrol . The genetic organization and regulation of these processes is also examined . This information is necessary for attempts aimed at the improvement of strains based on deregulating pathways or introducing traits from one strain to another . The release of genetically engineered organisms into the environment is governed by regulations, and this aspect is summarized . The commercialization of fluorescent pseudomonads for the biological control of plant root diseases remains an exciting possibility . The understanding of the relevant characteristics will facilitate this process by enabling the direct selection and/or construction of strains which will perform under a variety of environmental conditions.

J Med Genet, 1992 Dec, 29(12), 883 - 7
Severity of chest disease in cystic fibrosis patients in relation to their genotypes; al-Jader LN et al.; A detailed comparison of the severity of chest disease with mutational status was carried out by cross sectional study of 127 cystic fibrosis patients, aged 1 to 31 years, living in Wales . Lung disease was classified according to severity, depending on pulmonary function tests (carried out on 76 patients) and chest radiograph status; information was obtained also on age at diagnosis in relation to severity of chest disease and colonisation with Pseudomonas species . Genotypes were determined by analysis for the mutations delta F508, delta I507, G551D, R553X, G542X, R117H, R560T, 1717--IG > A, and 621 + 1G > T . CF patients homozygous positive and heterozygous for the delta F508 deletion showed a significant decline of lung function with age . Unlike other studies, we did not find patients homozygous positive for the delta F508 deletion to have poorer lung function compared with heterozygous patients . Patients with the genotype 621 + IG > T/delta F508 tended to have more severe chest disease than the delta F508 homozygous patients in the same age group . There was some evidence that four patients heterozygous for R117H have mild chest disease.

Appl Environ Microbiol, 1992 Dec, 58(12), 4068 - 71
Cloning and sequence analysis of genes for dehalogenation of 4-chlorobenzoate from Arthrobacter sp . strain SU; Schmitz A et al.; Strains of Arthrobacter catalyze a hydrolytic dehalogenation of 4-chlorobenzoate (4-CBA) to p-hydroxybenzoate . The reaction requires ATP and coenzyme A (CoA), indicating activation of the substrate via a thioester, like that reported for Pseudomonas sp . strain CBS3 (J . D . Scholten, K.-H . Chang, P . C . Babbit, H . Charest, M . Sylvestre, and D . Dunaway-Mariano, Science 253:182-185, 1991) . The dehalogenase genes of Arthrobacter sp . strain SU were cloned and expressed in Escherichia coli . Analyses of deletions indicate that dehalogenation depends on three open reading frames (ORFs) which are organized in an operon . There is extensive sequence homology to corresponding gene products in Pseudomonas sp . strain CBS3, suggesting that ORF1 and ORF2 encode a 4-CBA-CoA-ligase and a 4-CBA-CoA dehalogenase, respectively . ORF3 possibly represents a thioesterase, although no homology to the enzyme from Pseudomonas sp . strain CBS3 exists.

Appl Environ Microbiol, 1992 Dec, 58(12), 3879 - 82
Biphenomycin A production by a mixed culture; Ezaki M et al.; Production of biphenomycin A by Streptomyces griseorubiginosus 43708 was stimulated by a mixed culture with a partner strain, Pseudomonas maltophilia 1928 . This stimulatory effect on biphenomycin A accumulation by the mixed culture was caused by the enzyme activity which strain 1928 possessed . It is suggested that in a mixed culture strain 43708 produces a precursor of biphenomycin A in culture broth and that strain 1928 converts the precursor to biphenomycin A.

Appl Environ Microbiol, 1992 Dec, 58(12), 3873 - 8
Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain; Fenton AM et al.; Tn5 mutagenesis and complementation analysis were used to clone a 6-kb genomic fragment required for biosynthesis of 2,4-diacetylphloroglucinol (Phl) from fluorescent Pseudomonas sp . strain F113 . A recombinant plasmid, pCU203, containing this region partially complemented a Phl production-negative mutant (F113G22) derived from strain F113 . When sugar beet seeds were sown into an unsterilized soil, in which sugar beet was subject to damping-off by Pythium ultimum, the emergence of sugar beet seeds inoculated with strain F113 was significantly greater than that of seeds inoculated with F113G22 . Transfer of pCU203 into eight other Pseudomonas strains conferred the ability to synthesize Phl in only one of these strains, Pseudomonas sp . strain M114 . Strain M114(pCU203) showed enhanced antagonism towards P . ultimum in vitro and significantly increased the emergence of sugar beet seeds in the same soil compared with emergence induced by the parent strain M114.

Appl Environ Microbiol, 1992 Dec, 58(12), 3787 - 91
Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues; Frenken LG et al.; The lipA gene encoding the extracellular lipase produced by Pseudomonas glumae PG1 was cloned and characterized . A sequence analysis revealed an open reading frame of 358 codons encoding the mature lipase (319 amino acids) preceded by a rather long signal sequence of 39 amino acids . As a first step in structure-function analysis, we determined the Ser-Asp-His triad which makes up the catalytic site of this lipase . On the basis of primary sequence homology with other known Pseudomonas lipases, a number of putative active site residues located in conserved areas were found . To determine the residues actually involved in catalysis, we constructed a number of substitution mutants for conserved Ser, Asp, and His residues . These mutant lipases were produced by using P . glumae PG3, from which the wild-type lipase gene was deleted by gene replacement . By following this approach, we showed that Ser-87, Asp-241, and His-285 make up the catalytic triad of the P . glumae lipase . This knowledge, together with information on the catalytic mechanism and on the three-dimensional structure, should facilitate the selection of specific modifications for tailoring this lipase for specific industrial applications.

J Steroid Biochem Mol Biol, 1992 Dec, 43(7), 665 - 75
Homologies between enzymes involved in steroid and xenobiotic carbonyl reduction in vertebrates, invertebrates and procaryonts; Oppermann UC et al.; Evidence is reported for the existence of a structurally and functionally related and probably evolutionarily conserved class of membrane-bound liver carbonyl reductases/hydroxysteroid dehydrogenases involved in steroid and xenobiotic carbonyl metabolism . Carbonyl reduction was investigated in liver microsomes of 8 vertebrate species, as well as in insect larvae total homogenate and in purified 3 alpha-hydroxysteroid dehydrogenase preparations of the procaryont Pseudomonas testosteroni, using the ketone compound 2-methyl-1,2 di-(3-pyridyl)-1-propanone (metyrapone) as substrate . The enzyme activities involved in the metyrapone metabolism were screened for their sensitivity to several steroids as inhibitors . In all fractions tested, steroids of the adrostane or pregnane class strongly inhibited xenobiotic carbonyl reduction, whereas only in the insect and procaryotic species could ecdysteroids inhibit this reaction . Immunoblot analysis with antibodies against the respective microsomal mouse liver metyrapone reductase revealed strong crossrections in all fractions tested, even in those of the insect and the procaryont . A similar crossreaction pattern was achieved when the same fractions were incubated with antibodies against 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni . The mutual immunoreactivity of the antibody species against proteins from vertebrate liver microsomes, insects and procaryonts suggests the existence of structural homologies within these carbonyl reducing enzymes . This is further confirmed by limited proteolysis of purified microsomal mouse liver carbonyl reductase and subsequent analysis of the peptide fragments with antibodies specifically purified by immunoreactivity against this respective crossreactive antigen . These immunoblot experiments revealed a 22 kDa peptide fragment which was commonly recognized by all antibodies and which might represent a conserved domain of the enzyme.

J Pediatr Surg, 1992 Dec, 27(12), 1614 - 5
Intrauterine perineal tear: a rare birth injury; Bhat BV et al.; A rare case of birth injury having intrauterine complete perineal tear is presented . Defunctioning sigmoid colostomy was undertaken because of bad perineal condition . The baby died of Pseudomonas septicemia on the 15th day before definitive surgical procedure could be undertaken.

EMBO J, 1992 Dec, 11(13), 4677 - 84
Rapid activation of a novel plant defense gene is strictly dependent on the Arabidopsis RPM1 disease resistance locus; Kiedrowski S et al.; We cloned and sequenced cDNAs encoded by a novel plant defense gene, ELI3, from parsley and Arabidopsis thaliana . The predicted product shares no homology to known sequences . ELI3 mRNA accumulates in A . thaliana leaves in response to challenge with phytopathogenic Pseudomonas syringae strains . The timing and magnitude of this response are dictated by the genetics of the plant-pathogen interaction being analyzed . During incompatible interactions, where resistance in the plant genotype Col-0 is dictated by the dominant RPM1 locus, ELI3 mRNA accumulates to high levels 5-10 h post-inoculation . This kinetic behavior is also generated by the presence of a cloned bacterial avirulence gene, in otherwise virulent bacteria, which triggers resistance mediated via RPM1 action . The phenotypic outcome is a hypersensitive resistance reaction visible 8-15 h post-infiltration . Thus, the induction kinetics of ELI3 mRNA accumulation are consistent with a functional role for the ELI3 gene product in establishing the resistant phenotype . In contrast, during compatible interactions with the susceptible plant genotype Nd-0, which is homozygous recessive at the rpm1 locus, ELI3 mRNA accumulates significantly only after 15 h . We show genetically that ELI3 activation is strictly dependent on the presence of dominant alleles at RPM1 using an assay generalizable to any pathogen induced plant defense phenomena.

J Bacteriol, 1992 Dec, 174(24), 7989 - 95
Purification and characterization of the hydantoin racemase of Pseudomonas sp . strain NS671 expressed in Escherichia coli; Watabe K et al.; The hydantoin racemase gene of Pseudomonas sp . strain NS671 had been cloned and expressed in Escherichia coli . Hydantoin racemase was purified from the cell extract of the E . coli strain by phenyl-Sepharose, DEAE-Sephacel, and Sephadex G-200 chromatographies . The purified enzyme had an apparent molecular mass of 32 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . By gel filtration, a molecular mass of about 190 kDa was found, suggesting that the native enzyme is a hexamer . The optimal conditions for hydantoin racemase activity were pH 9.5 and a temperature of 45 degrees C . The enzyme activity was slightly stimulated by the addition of not only Mn2+ or Co2+ but also metal-chelating agents, indicating that the enzyme is not a metalloenzyme . On the other hand, Cu2+ and Zn2+ strongly inhibited the enzyme activity . Kinetic studies showed substrate inhibition, and the Vmax values for D- and L-5-(2-methylthioethyl)hydantoin were 35.2 and 79.0 mumol/min/mg of protein, respectively . The purified enzyme did not racemize 5-isopropylhydantoin, whereas the cells of E . coli expressing the enzyme are capable of racemizing it . After incubation of the purified enzyme with 5-isopropylhydantoin, the enzyme no longer showed 5-(2-methylthioethyl)hydantoin-racemizing activity . However, in the presence of 5-(2-methylthioethyl)hydantoin, the purified enzyme racemized 5-isopropylhydantoin completely, suggesting that 5-(2-methylthioethyl)hydantoin protects the enzyme from inactivation by 5-isopropylhydratoin . Thus, we examined the protective effect of various compounds and found that divalent-sulfur-containing compounds (R-S-R' and R-SH) have this protective effect.

Genetika, 1992 Dec, 28(12), 31 - 7
{Cloning the trpBA-genes from Pseudomonas mendocina and Pseudomonas marginata}; Olekhnovich IN et al.; The trpBA genes of Pseudomonas mendocina and P . marginata were cloned in Escherichia coli using pBR322 as a vector . Transcription of the cloned genes took place from their own promoters, in the following order: trpB-->trpA . The latter genes were not linked to the trpF gene and could not be induced by indole glycerol phosphate . Thus, the trpBA cluster of P . mendocina and P . marginata are distinguished from that of P . putida, P . aeruginosa (trpIBA) and P . acidovorans (trpFBA).

Biotechnol Appl Biochem, 1992 Dec, 16(3), 228 - 35
The use of detergent-based aqueous two-phase systems for the isolation of extracellular proteins: purification of a lipase from Pseudomonas cepacia; Terstappen GC et al.; The partitioning of a variety of extracellular lipases, both pro- and eucaryotic, in detergent-based aqueous two-phase systems was examined . The results revealed that all procaryotic lipases showed a clear preference for the detergent-rich coacervate phase . In contrast, all eucaryotic lipases were significantly excluded from this phase, most probably caused by their glycosylation . The potential of such detergent-based systems for the isolation of extracellular lipases directly from cell-free culture broth was analyzed using the bacterium Pseudomonas cepacia (DSM 50181) . This strain was identified after a limited screening for lipase activity . About 76% of the lipase could be extracted into the coacervate phase in just one purification step, leading to a four-fold concentration of lipase and a purification factor of 24.

Appl Environ Microbiol, 1992 Dec, 58(12), 3977 - 83
Selection of a Pseudomonas cepacia strain constitutive for the degradation of trichloroethylene; Shields MS et al.; Tn5 insertion mutants of Pseudomonas cepacia G4 that were unable to degrade trichloroethylene (TCE), toluene, or phenol or to transform m-trifluoromethyl phenol (TFMP) to 7,7,7-trifluoro-2-hydroxy-6-oxo-2,4-heptadienoic acid (TFHA) were produced . Spontaneous reversion to growth on phenol or toluene as the sole source of carbon was observed in one mutant strain, G4 5223, at a frequency of approximately 1 x 10(-4) per generation . One such revertant, G4 5223-PR1, metabolized TFMP to TFHA and degraded TCE . Unlike wild-type G4, G4 5223-PR1 constitutively metabolized both TFMP and TCE without aromatic induction . G4 5223-PR1 also degraded cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, and 1,1-dichloroethylene and oxidized naphthalene to alpha naphthol constitutively . G4 5223-PR1 exhibited a slight retardation in growth rate at TCE concentrations of > or = 530 microM, whereas G4 (which was unable to metabolize TCE under the same noninducing growth conditions) remained unaffected . The constitutive degradative phenotype of G4 5223-PR1 was completely stable through 100 generations of nonselective growth.

J Biol Chem, 1992 Nov 25, 267(33), 24034 - 40
Pseudomonas exotoxin A-epidermal growth factor (EGF) mutant chimeric protein as an indicator for identifying amino acid residues important in EGF-receptor interaction; Shiah HS et al.; Epidermal growth factor (EGF) was fused to the carboxyl end of a modified pseudomonas exotoxin A that has its toxin binding domain deleted . This chimeric toxin designated as PE(delta Ia)-EGF kills A431 cells through the EGF receptor-mediated pathway . In this study, we used a random mutagenesis approach to make point mutations on EGF, followed by replacing the wild type EGF in PE(delta Ia)-EGF with these EGF mutants . We have constructed 14 different PE(delta Ia)-EGFmutants, and examined their EGF receptor binding activity as well as their cytotoxicity to A431 cells . Our results showed that individual mutations of Val19 to Glu and Val34 to Asp in the EGF domain of PE(delta Ia)-EGFmutants resulted in an increase in the binding affinity to EGF receptor and cytotoxicity to A431 cells . On the other hand, individual mutations of His16 to Asp and Gly18 to Ala in the EGF domain of PE(delta Ia)-EGFmutants lead to a decrease in the binding affinity to EGF receptor and cytotoxicity to A431 cells . In addition, mutations of any of the cysteine residues of EGF in PE(delta Ia)-EGFmutants resulted in the loss of their binding activity to EGF receptor and a corresponding loss of their cytotoxicity . This study indicates that the cytotoxicity of PE(delta Ia)-EGFmutant to EGF receptor-bearing cells may be used as an indicator to screen mutations of EGF important in EGF-receptor interactions.

J Biol Chem, 1992 Nov 15, 267(32), 23427 - 33
Alanine scanning mutagenesis identifies surface amino acids on domain II of Pseudomonas exotoxin required for cytotoxicity, proper folding, and secretion into periplasm; Kasturi S et al.; Pseudomonas exotoxin A (PE) is a single polypeptide chain that contains 613 amino acids and is arranged into three major structural domains . Domain Ia is responsible for cell recognition, domain II for translocation of PE across the membrane, and domain III for ADP-ribosylation of elongation factor 2 . Recombinant PE can be produced in Escherichia coli and is efficiently secreted into the periplasm when an OmpA signal sequence is present . To investigate the role of the amino acids located on the surface of domain II in the action of the toxin against mammalian cells, we substituted alanine for each of the 27 surface amino acids present in domain II . Surprisingly, all 27 mutant proteins had some alteration in cytotoxicity when tested on human A431 or MCF7 cells or mouse L929 cells . Native PE has a compact structure and therefore is relatively protease resistant and very little ADP-ribosylation activity is detected in the absence of the denaturing agents like urea and dithiothreitol . Several of the mutations resulted in altered protease sensitivity of the toxin . Seven of the mutant molecules exhibited ADP-ribosylation activity without urea and dithiothreitol, indicating they are partially unfolded . Out of these seven mutants, six had increased cytotoxic activity on at least one of the target cell lines and the other retained its native cytotoxic potency.

J Biol Chem, 1992 Nov 15, 267(32), 22897 - 901
Identification of critical amino-terminal regions of XylS . The positive regulator encoded by the TOL plasmid; Michan C et al.; The XylS protein is the positive regulator of the promoter controlling the meta-cleavage pathway (Pm) for catabolism of certain alkylbenzoates on the TOL plasmid of Pseudomonas . Transcription from Pm is mediated by XylS either in the presence of benzoate effectors or through XylS hyperproduction . Two regions of the NH2 terminus of XylS (residues 37-45) had been predicted to be involved in effector control of XylS transcriptional activation . Different methods were used to induce mutations in this region, including genetic selections (where Pm controlled a tetracycline resistance gene), bisulfite mutagenesis at a unique restriction site, and extensive oligonucleotide mutagenesis at residues 41 and 45 . The mutants fell into four classes based on their phenotypes with respect to effector-mediated activation of a Pm-lacZ fusion: (a) effector profiles similar to wild type, (b) no Pm stimulation with benzoates, (c) altered effector specificity, and (d) higher basal Pm activities, in some cases including changes in effector specificity . In some mutants, higher basal Pm activity was apparently due to mutations that increased XylS stability . Substitutions at Arg-41 resulted in all four mutant phenotypes, indicating that this is a critical residue in XylS for effector stimulation of transcription activity.

FEBS Lett, 1992 Nov 2, 312(1), 3 - 6
The second cholera toxin, Zot, and its plasmid-encoded and phage-encoded homologues constitute a group of putative ATPases with an altered purine NTP-binding motif; Koonin EV; It is shown that the second cholera toxin, Zot, ORF3 product of Pseudomonas plasmid pKB740, and ORF424 product of bacteriophage Pf1 are a group of closely related proteins containing a modified version of the purine NTP-binding motif, with a drastic substitution of tyrosine for a conserved glycine . They are distantly but reliably related to the product of gene I of filamentous bacteriophages which is a putative ATPase containing the classical NTP-binding motif and is involved in bacteriophage assembly and exit from the bacterial cell . Hydropathy analysis suggests that the Zot and gene I product may have a similar transmembrane topology . It is hypothesized that Zot may possess ATPase activity and modify the membrane structure of its target cells in an ATP-dependent fashion . Genes for Zot and the related protein of pKB740 are likely to have evolved from gene I of a Pf1-like bacteriophage.

Antimicrob Agents Chemother, 1992 Nov, 36(11), 2512 - 7
Increased oral bioavailability of ciprofloxacin in cystic fibrosis patients; Christensson BA et al.; The altered pharmacokinetic properties of, e.g., aminoglycosides in cystic fibrosis patients have to be considered when pulmonary exacerbations are treated . Since reported data on ciprofloxacin, a fluorinated quinolone, are conflicting, we compared intravenous and oral administration in cystic fibrosis patients when treating them for mild symptoms of pulmonary infection . All of the patients were colonized with Pseudomonas species . Ciprofloxacin was administered orally (15 mg/kg of body weight) or intravenously (6 mg/kg) twice a day for at least 10 days during separate treatment periods . Five healthy volunteers received single intravenous and oral doses . Pharmacokinetic evaluations were performed at first dose and at steady state . The results showed that cystic fibrosis patients have increased oral bioavailability of ciprofloxacin (80% in cystic fibrosis patients versus 57% in volunteers) and increased total clearance (688 ml/min in CF patients versus 528 ml/min in volunteers) . Our data indicate that the pharmacokinetic properties of ciprofloxacin are altered in cystic fibrosis patients with mild symptoms of pulmonary exacerbations and that the changes most probably are due to cystic fibrosis per se or to the impact of chronic infection.

Appl Environ Microbiol, 1992 Nov, 58(11), 3759 - 61
Use of tRNA consensus primers to indicate subgroups of Pseudomonas solanacearum by polymerase chain reaction amplification; Seal SE et al.; Polymerase chain reaction amplification of DNA from 112 Pseudomonas solanacearum strains with the tRNA consensus primers T3A and T5A divided the species into three fingerprint groups . These groups correspond well with previous divisions made by restriction fragment length polymorphism analysis . This polymerase chain reaction test is a facile method for rapidly classifying P . solanacearum strains.

Appl Environ Microbiol, 1992 Nov, 58(11), 3751 - 8
Isolation of a Pseudomonas solanacearum-specific DNA probe by subtraction hybridization and construction of species-specific oligonucleotide primers for sensitive detection by the polymerase chain reaction; Seal SE et al.; A subtraction hybridization technique was employed to make a library enriched for Pseudomonas solanacearum-specific sequences . One cloned fragment, PS2096, hybridized under stringent conditions to DNA of 82 P . solanacearum strains representing all subgroups of the species . Other plant-associated bacteria, including closely related species such as Pseudomonas capacia, Pseudomonas picketti, or Pseudomonas syzygii, did not hybridize to PS2096 . A minimum number of between 4 x 10(5) and 4 x 10(6) P . solanacearum cells could routinely be detected with PS2096 labelled either with {32P}dCTP or with digoxigenin-11-dUTP . To improve the sensitivity of detection, PS2096 was sequenced to allow the construction of specific oligonucleotide primers to be used for polymerase chain reaction (PCR) amplification . After 50 cycles of amplification, 5 to 116 cells, depending on the strain, could reproducibly be detected by visualization of a 148-bp PCR product on an agarose gel . A preliminary field trial in Burundi with the probe and PCR primers has confirmed that they are sensitive tools for specifically detecting low-level infections of P . solanacearum in potato tubers.

Plant Cell, 1992 Nov, 4(11), 1359 - 69
Functional homologs of the Arabidopsis RPM1 disease resistance gene in bean and pea; Dangl JL et al.; We showed that a bacterial avirulence (avr) gene function, avrPpiA1, from the pea pathogen Pseudomonas syringae pv pisi, is recognized by some, but not all, genotypes of Arabidopsis . Thus, an avr gene functionally defined on a crop species is also an avr gene on Arabidopsis . The activity of avrPpiA1 on a series of Arabidopsis genotypes is identical to that of the avrRpm1 gene from P.s . pv maculicola previously defined using Arabidopsis . The two avr genes are homologous and encode nearly identical predicted products . Moreover, this conserved avr function is also recognized by some bean and pea cultivars in what has been shown to be a gene-for-gene manner . We further demonstrated that the Arabidopsis disease resistance locus, RPM1, conditioning resistance to avrRpm1, also conditions resistance to bacterial strains carrying avrPpiA1 . Therefore, bean, pea, and conceivably other crop species contain functional and potentially molecular homologs of RPM1.

Br J Anaesth, 1992 Nov, 69(5), 522 - 5
Bacterial retention properties of heat and moisture exchange filters; Lee MG et al.; We have examined the properties of six heat and moisture exchange filters (HMEF) to ascertain their resistance to liquid flow and their ability to retain a challenge bacterium, Pseudomonas diminuta, from aqueous and nebulized suspensions . Only one HMEF, the Pall Ultipor was able to withstand a significantly greater pressure of liquid than that found in clinical practice . However, when breached, the HMEF were unable to prevent transmission of micro-organisms from aqueous suspension . Only the Darex Hydrobac filter failed to meet the manufacturer's claim for filter efficiency for nebulized bacteria, mainly because the filter housing failed under test . When the reduction in bacterial cells after passage of the nebulized Pseudomonas diminuta through the HMEFs was analysed statistically, the data showed that the HMEF produced by Pall (Ultipor) and Intersurgical (Filter therm) were superior to those produced by DAR Mediplan (Hygrobac), Intertech (HME 225-2835-800) and Gibeck (Humid-vent).

Prof Nurse, 1992 Nov, 8(2), 100 - 4
An opportunist to keep at bay . Pseudomonas in ITU; Woodrow P; Pseudomonas is a common cause of infection in ITU . To minimise incidence of this opportunistic infection, it is essential that nursing care is based on research, rather than ritual.

Blood, 1992 Nov 1, 80(9), 2344 - 52
Recombinant toxins containing the variable domains of the anti-Tac monoclonal antibody to the interleukin-2 receptor kill malignant cells from patients with chronic lymphocytic leukemia; Kreitman RJ et al.; We have previously shown that the variable domains of the monoclonal antibody anti-Tac {anti-Tac(Fv)} can be fused to derivatives of Pseudomonas exotoxin (PE) or diphtheria toxin (DT) to produce recombinant immunotoxins that kill interleukin-2 (IL-2) receptor-bearing cells . We now report that two of these single-chain recombinant immunotoxins, anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv), are cytotoxic toward peripheral blood mononuclear cells (PBMCs) from patients with chronic lymphocytic leukemia (CLL) . In anti-Tac(Fv)-PE40KDEL, anti-Tac(Fv) is genetically fused to the amino terminus of PE40KDEL, a recombinant form of PE which contains amino acids 253-608 of PE and the -KDEL mutation at the carboxyl terminus . In DT388-anti-Tac(Fv), anti-Tac(Fv) is fused to the carboxyl terminus of the first 388 amino acids of DT . PBMCs from 14 patients were incubated with the recombinant toxins for 60 hours, and {3H}-leucine incorporation was measured . Anti-Tac(Fv)-PE40KDEL was cytotoxic to 7 of the 14 patient samples, with half-maximal inhibition of protein synthesis (IC50) achieved at 1.2 to 9 ng/mL (1.8 to 13 x 10(-11) mol/L) . DT388-anti-Tac(Fv) was cytotoxic to 11 of the 14 samples, with IC50s ranging from less than 1 to 250 ng/mL . DT388-IL-2, in which the first 388 amino acids of DT are attached to IL-2, was marginally cytotoxic toward only 4 of 13 CLL samples tested with IC50s ranging from 100 to 550 ng/mL . Trypan blue staining of cells from several patients indicated that inhibition of protein synthesis correlated with cell death . Binding assays using {3H}-anti-Tac indicated that the CLL cells from nine of the patients contained between 400 and 2,500 sites per cell . Cells from another patient, which were resistant to both anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv), had less than 100 sites per cell . We conclude that anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv) can kill CLL cells which have low numbers of IL-2 receptors, and should be investigated further for therapy of this disease.

Genetika, 1992 Nov, 28(11), 34 - 9
{Plasmid determined degradation of sulfo-aromatic compounds by Pseudomonas sp . BS1304}; Dubeikovskii AN et al.; Utilized sulfo-aromatic compounds of Pseudomonas sp . BS1304 were isolated . It was shown that the first step of conversion is desulfonation with following meta-cleavage of the substituted aromatic ring . At least two steps are controlled by the plasmid pBS1004 (120 kb), belonging to the IncP-9 incompatibility group . The degradative plasmid marked by Tn5-lac may be mobilized to P . putida, P . aeruginosa, P . mendocina, where a degradative phenotype is expressed.

J Exp Biol, 1992 Nov, 172, 245 - 66
Chloride channels of intracellular organelles and their potential role in cystic fibrosis; al-Awqati Q et al.; Chloride channels were previously purified from bovine kidney cortex membranes using a drug affinity column . Reconstitution of the purified proteins into artificial liposomes and planar bilayers yielded chloride channels . A 64 x 10(3) M(r) protein, p64, identified as a component of this chloride channel, was used to generate antibodies which depleted solubilized kidney membranes of all chloride channel activity . This antibody has now been used to identify a clone, H2B, from a kidney cDNA library . Antibodies, affinity-purified against the fusion protein of H2B, from a kidney cDNA library . Antibodies, affinity-purified against the fusion protein of H2B, also depleted solubilized kidney cortex from all chloride channel activity . The predicted amino acid sequence of p64 shows that it contains two and possibly four putative transmembrane domains and potential phosphorylation sites by protein kinases A and C . There was no significant homology to other protein (or DNA) sequences in the data base including other anion channels or the cystic fibrosis transmembrane conductance regulator . The protein is expressed in all cells tested and probably represents the chloride channel of intracellular organelles . Cystic fibrosis (CF) is associated with a defect in a cyclic-AMP-activated chloride channel in secretory epithelia which leads to decreased fluid secretion . In addition, many mucus glycoproteins show decreased sialylation but increased sulfation . We have recently shown that the pH of intracellular organelles is more alkaline in CF cells, an abnormality that is due to defective chloride conductance in the vesicle membranes . We postulate that the defect in the intracellular chloride channel, and hence the alkalization, could explain the glycosylation abnormalities since the pH optimum of Golgi sialyltransferase is acid while that of focusyl- and sulfotransferases is alkaline . Defects in sialyation of glycolipids might also generate receptors for Pseudomonas, which is known to colonize the respiratory tract of CF patients.

Infection, 1992 Nov-Dec, 20(6), 367 - 8
Pseudomonas vesicularis bacteraemia; Planes AM et al.; A case of repeated episodes of Pseudomonas vesicularis bacteraemia, in a 54-year-old woman with a past history including systemic lupus erythematosus and chronic active autoimmune hepatitis is reported . She was treated with tobramycin and ceftazidime but bacteraemia persisted until surgical resection of the infected tissue was performed.

Mikrobiol Zh, 1992 Nov-Dec, 54(6), 54 - 8
{The effect of the acid exopolysaccharide produced by the causative agent of melioidosis on macrophage function}; Bozhko VG et al.; Acid exopolysaccharide of the melioidosis agent, isolated from the slime of alysogenic strain of Pseudomonas pseudomallei SVBDO-141 inhibits the capture of sheep erythrocytes--55Fe by mouse peritoneal macrophages . Administration of the sheep erythrocytes--55Fe, sensibilized by acid exopolysaccharide was proved not to have an antiphagocytic effect . Inhibition of phagocytic macrophagal activity depends on the conditions of isolation of acid exopolysaccharide.

J Biochem (Tokyo), 1992 Nov, 112(5), 598 - 603
Purification and characterization of a novel thermostable lipase from Pseudomonas cepacia; Sugihara A et al.; A thermostable lipase from Pseudomonas cepacia has been purified to homogeneity as judged by SDS-PAGE and isoelectric focusing . The purification included treatment of the culture supernatant with acrinol, hydrophobic interaction chromatography, and gel filtration . The enzyme was a monomeric protein with M(r) of 36,500 and pI of 5.1 . The optimal pH at 50 degrees C and optimal temperature at pH 6.5 were 5.5-6.5 and 55-60 degrees C, respectively, when olive oil was used as the substrate . Simple triglycerides of short and middle chain fatty acids (C < or = 12) were the preferred substrates over those of long chain fatty acids . The enzyme cleaved all the ester bonds of triolein, with some preference for the 1,3-ester bonds . The enzyme retained all its activity even after incubation at 75 degrees C (pH 6.5) for 30 min . Further, the activity was not impaired during 21 h storage at pH 6.5 in 40% water-miscible solvents including methanol, ethanol, acetone, acetonitrile, dimethylformamide, dimethylsulfoxide, and dioxane . The addition of dimethylsulfoxide or acetone to the assay mixture in the range of 0-35% stimulated the enzyme, whereas benzene or n-hexane had an inhibitory effect . These properties together with the N-terminal amino acid sequence confirmed that the enzyme differs from the known Pseudomonas sp . lipases.

J Med Microbiol, 1992 Nov, 37(5), 335 - 40
Inhibition of rat alveolar macrophage phagocytic function by a Pseudomonas cepacia lipase; Straus DC et al.; The effects of purified Pseudomonas cepacia lipase on rat pulmonary alveolar function and morphology were examined . Lipase (2.5-20 micrograms/ml) adversely effected the phagocytic function of rat pulmonary alveolar macrophages in a dose-dependent manner . The lipase itself was not directly cytotoxic to these cells . Alveolar macrophages, in the absence of lipase, phagocytosed c . 35% of a given population of opsonised P . cepacia in 30 min when the ratio of bacteria:phagocyte was 10:1 . Phagocytosis of P . cepacia by rat pulmonary alveolar macrophages was significantly reduced when the cells were either pre-incubated with the lipase or when phagocytosis occurred in the presence of the lipase . This was confirmed by transmission electronmicroscopy . These functional changes were associated with marked alterations of the macrophage morphology . Scanning electronmicroscopy showed that macrophages exposed to the P . cepacia lipase had fewer specialised surface structures and did not spread on plastic surfaces as well as untreated macrophages . The effects of the lipase were lost after heat inactivation, which indicates that the effects of the P . cepacia lipase were due to its enzymic activity . These results suggest that, if sufficient quantities of the enzyme are produced in vivo, lipase may be an important virulence factor for P . cepacia, allowing the organism to evade phagocytic cells.

Biochem Biophys Res Commun, 1992 Oct 30, 188(2), 826 - 32
Molecular cloning in Escherichia coli, expression, and nucleotide sequence of the gene for the ethylene-forming enzyme of Pseudomonas syringae pv . phaseolicola PK2; Fukuda H et al.; The gene for the ethylene-forming enzyme of Pseudomonas syringae pv . phaseolicola PK2 was found to be encoded by an indigenous plasmid, designated pPSP1 . The gene for the ethylene-forming enzyme was cloned and expressed in Escherichia coli JM109 . Nucleotide sequence analysis of the clone revealed an open reading frame that encodes 350 amino acids (mol . wt . 39,444) . In a comparison with other proteins, the homology score for the entire amino-acid sequence of the ethylene-forming enzyme of Pseudomonas syringae versus ethylene-forming enzymes from plants and 2-oxoglutarate-dependent dioxygenases was low . However, functionally significant regions are conserved.

Biochem Biophys Res Commun, 1992 Oct 30, 188(2), 483 - 9
Two reactions are simultaneously catalyzed by a single enzyme: the arginine-dependent simultaneous formation of two products, ethylene and succinate, from 2-oxoglutarate by an enzyme from Pseudomonas syringae; Fukuda H et al.; A single enzyme isolated from Pseudomonas syringae pv . phaseolicola PK2 simultaneously catalyzed two reactions, namely, the formation of ethylene and succinate from 2-oxoglutarate, at a molar ratio of 2:1 . In the main reaction, 2-oxoglutarate was dioxygenated to produce one molecule of ethylene and three molecules of carbon dioxide . In the sub-reaction, both 2-oxoglutarate and L-arginine were mono-oxygenated to yield succinate plus carbon dioxide and L-hydroxyarginine, respectively, the latter being further transformed to guanidine and L-delta 1-pyrroline-5-carboxylate . We propose a dual-circuit mechanism for the entire reaction, in which the binding of L-arginine and 2-oxoglutarate in a Schiff-base structure generates a common intermediate for two reactions.

J Am Vet Med Assoc, 1992 Oct 15, 201(8), 1229 - 32
Ocular manifestation of systemic histiocytosis in a dog; Scherlie PH Jr et al.; A 3-year-old sexually intact female Bernese Mountain Dog was referred for treatment of a descemetocele of the left eye . Physical examination revealed bilateral exophthalmos and scleral thickening, multiple cutaneous nodules, and mandibular paralysis in addition to ulcerative keratitis associated with a Pseudomonas infection . One year prior to examination, a biopsy specimen of the episcleral tissue from the right eye had been interpreted as nodular granulomatous episclerokeratitis (fibrous histiocytoma) . Immunosuppressive treatment prior to referral had not resulted in remission of the ocular lesions . When we examined the biopsy specimen, we interpreted the lesions to represent episcleral involvement of systemic histiocytosis . Because of the poor prognosis, the dog was euthanatized.

Int J Cancer, 1992 Oct 21, 52(4), 631 - 5
Immunotoxins directed against the high-molecular-weight melanoma-associated antigen . Identification of potent antibody-toxin combinations; Godal A et al.; To study factors influencing the cytotoxicity of immunotoxins (ITs), we compared the in vitro cytotoxicity of conjugates in which the plant toxin abrin and the bacterial toxin Pseudomonas exotoxin A (PE) were coupled by 2 different procedures to 2 MAbs, 9.2.27 and NR-ML-05, which bind to different epitopes on the melanoma-associated antigen p250 . The individual target cell lines differed widely in sensitivity to the different ITs, as assessed by measurement of protein synthesis inhibition . The action of the ITs was highly specific, as the toxicity of abrin and PE conjugates was respectively 20-540 and 2,200-550,000 times higher in antigen-positive cell lines (FEMX, SESX, OHS) than in the antigen-negative line KPDX . The PE conjugates prepared with the 2 different MAbs differed in potency by factors of 16-126 in the target-cell lines, but were consistently more toxic than the abrin ITs . The results demonstrate that the cytotoxicity of ITs varies with the nature of both of its moieties and that optimal results require that toxins and MAbs be matched . Moreover, the 2 coupling procedures affected differentially the binding and potency of some ITs . Each of the 2 toxins was conjugated to a sheep anti-mouse antibody (SAM) and the toxicity of these 2 conjugates was tested in an indirect approach using 9.2.27 and NR-ML-05 as primary MAbs . The results showed that the indirect procedure would have correctly predicted the most potent antibody-toxin pair, indicating that the approach may be valid for selecting suitable combinations of MAbs and toxins for use as direct ITs.

Biochim Biophys Acta, 1992 Oct 20, 1132(3), 233 - 9
Nucleotide sequence and expression in Escherichia coli of the cephalosporin acylase gene of a Pseudomonas strain; Ishiye M et al.; The gene encoding cephalosporin acylase, which hydrolyzes 7-beta-(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA) to 7-aminocephalosporanic acid (7ACA) and glutaric acid, was cloned from a Pseudomonas sp . strain V22 and expressed in Escherichia coli, in a two-cistron system, and the enzyme was purified and characterized . The purified enzyme was composed of two non-identical subunits, their molecular weights were estimated by SDS-PAGE to be 40,000 and 22,000, and had a pI of 4.6 . The amino acid sequence of the enzyme, deduced from the nucleotide sequence, showed high similarity (97%) with that of a previously reported acyI-encoded cephalosporin acylase . Cephalosporin acylase also resembles the bacterial gamma-glutamyl transpeptidases (GGTs) with respect to their molecular organization and amino acid sequence, but differs from them with respect to catalytic and immunological properties . Purified enzyme exhibited not only cephalosporin acylase activity, but also GGT activity . The Km values of the enzyme for GL-7ACA and L-gamma-glutamyl-p-nitroanilide were 6.1 and 3.8 mM, respectively . Cephalosporin acylase was not recognized by antibodies prepared against bacterial GGTs.

J Mol Biol, 1992 Oct 20, 227(4), 1258 - 62
Crystallization and preliminary X-ray crystallographic analysis of lipase from Pseudomonas cepacia; Kim KK et al.; Large crystals of lipase from Pseudomonas cepacia have been grown at room temperature from solutions containing 2-methyl-2,4-pentanediol and sodium citrate . They grow within two weeks to typical dimensions of 1.0 mm x 0.5 mm x 0.3 mm . The crystals belong to the monoclinic space group P2(1), with unit cell parameters a = 84.91 A, b = 47.33 A, c = 86.00 A, and beta = 116.09 degrees . And they diffract to about 1.6 A upon exposure to synchroton X-rays . X-ray data have been collected to 2.2 A Bragg spacing from a native crystal.

FEMS Microbiol Lett, 1992 Oct 15, 76(3), 261 - 6
Characterization of isofunctional ring-cleaving enzymes in aniline and 3-chloroaniline degradation by Pseudomonas acidovorans CA28; Hinteregger C et al.; During degradation of aniline and 3-chloroaniline, respectively, by Pseudomonas acidovorans CA28, selective induction of two catechol 1,2-dioxygenases (C12O) was observed . C12O I activity was the sole ring-cleaving enzyme detectable in cell-free extracts after growth on aniline, while C12O II was exclusively found after growth on 3-chloroaniline . Both enzymes were clearly differentiated by their elution behaviour on DEAE-cellulose and their substrate specificities . For C12O I high activity was demonstrable only with unsubstituted catechol, while C12O II showed preference for and high affinity towards chlorinated catechols . Therefore, evidence of different ortho-cleavage enzymes in Pseudomonas acidovorans CA28 involved in aniline and 3-chloroaniline metabolism, respectively, is indicated.

J Immunol, 1992 Oct 15, 149(8), 2810 - 5
Mik-beta 1(Fv)-PE40, a recombinant immunotoxin cytotoxic toward cells bearing the beta-chain of the IL-2 receptor; Kreitman RJ et al.; Mik-beta 1 is a mAb that binds to the beta subunit of the IL-2R . We have constructed a recombinant single chain immunotoxin Mik-beta 1(Fv)-PE40 by genetically fusing the H and L V domains of Mik-beta 1 to each other via a peptide linker, and then to PE40, a derivative of Pseudomonas exotoxin . Mik-beta 1(Fv)-PE40 was selectively cytotoxic for cells expressing high levels of IL-2R beta (p75) subunit . Mik-beta 1(Fv)-PE40 was cytotoxic to the NK cell line YT-S, which expresses p75 but not p55 subunits, with an IC50 of 6 ng/ml . The ATL line HUT-102 was less sensitive, with an IC50 of 200 ng/ml . However, the IC50 could be lowered to 11 ng/ml when Mik-beta 1(Fv)-PE40 was allowed to bind to HUT-102 cells at 4 degrees C for 4 h before overnight incubation at 37 degrees C . An excess of Mik-beta 1 but not of anti-Tac, the anti-p55 mAb, prevented the cytotoxicity of Mik-beta 1(Fv)-PE40 . We constructed a more active version of Mik-beta 1(Fv)-PE40, designated Mik-beta 1(Fv)-PE40KDEL, by converting the carboxyl-terminus of the toxin from -REDLK to -KDEL . Mik-beta 1(Fv)-PE40KDEL showed an IC50 of 2 ng/ml toward YT-S cells and 35 ng/ml toward HUT-102 cells . Binding studies using radioiodinated Mik-beta 1 showed that Mik-beta 1(Fv)-PE40 bound to the p75 receptor subunit with 11% of the affinity of the native Mik-beta 1 antibody . Mik-beta 1(Fv)-PE40 may be a useful reagent to study cells that express IL-2R, and it deserves further study as a possible treatment for cancers in which the malignant cells express high numbers of p75 subunit.

FEMS Microbiol Lett, 1992 Oct 15, 76(3), 235 - 41
Bacterial gamma-glutamyltranspeptidases: comparison of Escherichia coli and Pseudomonas gamma-glutamyltranspeptidase; Ishiye M et al.; A high-level expression system for the Escherichia coli HB101 gamma-glutamyltranspeptidase (GGT) gene was constructed to obtain a sufficient amount of the enzyme for structural studies . About 300 mg of the enzyme was purified from 2 1 of culture broth . Both subunits were needed to reconstitute GGT activity . The E . coli GGT and the Pseudomonas GGT were compared with respect to catalytic properties and immunological relationships . Both enzymes showed different acceptor specificities . Relatively high immunocross-reactivity was observed between the small subunits of both enzymes by Western blot analysis using antibodies prepared against either enzyme.

Curr Opin Oncol, 1992 Oct, 4(5), 946 - 54
Biology and therapy with biologic agents in gynecologic cancer; Wiener JR et al.; Growth of epithelial ovarian cancer is influenced by several factors including transforming growth factor-alpha and transforming growth factor-beta, macrophage colony stimulating factor, tumor necrosis factor-alpha, interleukin-1 and interleukin-6, c-erb B-2 (HER-2/neu), and mutant p53 . Continued expression of the epidermal growth factor receptor, new expression of c-fms, and overexpression of HER-2/neu are associated with a poor prognosis . A number of cytokines have been used to treat patients with ovarian cancer, including interferon-alpha, interferon-gamma, tumor necrosis factor-alpha, and interleukin-2 . Judging from preclinical models, interferon-gamma may be more active than interferon-alpha against human ovarian cancer . Although tumor necrosis factor-alpha can stimulate proliferation of some ovarian cancers, the cytotoxic activity of tumor necrosis factor-alpha has been amplified ex vivo by inhibitors of protein synthesis . Similar heterogeneity exists with regard to interleukin-1 where stimulation or inhibition of cell proliferation has been observed . Tumor-infiltrating lymphocytes from ascites fluid contain cells capable of major histocompatibility complex-restricted and major histocompatibility complex-nonrestricted cytotoxicity . Tumor-infiltrating lymphocytes and interleukin-2 have been combined with cytotoxic chemotherapy to treat advanced or recurrent disease . Bispecific monoclonal antibodies that react both with T cells and ovarian tumor cells have produced tumor inhibition in human tumor xenografts . Immunotoxins that contain OVB3 and pseudomonas exotoxin have been evaluated in a phase I clinical trial . Dose-limiting central neurotoxicity has been observed without tumor regression . A monoclonal antibody designated OVX1 has been developed against a high-molecular-weight mucinlike molecule associated with ovarian cancers.(ABSTRACT TRUNCATED AT 250 WORDS)

Virology, 1992 Oct, 190(2), 635 - 44
Bacteriophage phi 6 envelope elucidated by chemical cross-linking, immunodetection, and cryoelectron microscopy; Kenney JM et al.; Bacteriophage phi 6 is an enveloped dsRNA virus which infects the plant pathogenic Pseudomonas syringae bacterium . Using low dose cryoelectron microscopy we show that the nucleocapsid, spikeless virion, and intact virion have radii of 29, 35, and 43 nm, respectively . Thus, the membrane is 6 nm thick and the surface spikes of the receptor binding protein P3 extend 8 nm from the membrane surface . Cross-linking, immunological, and complementation evidence suggest that the spikes are formed of multimeric P3 molecules and that P3 is associated with membrane-bound protein P6 . We observe that the envelope can accommodate up to 400 molecules of P3 but that the average virion contains less than one-fourth of this amount . Assembly of a very small number of P3 or truncated P3 molecules onto inactive virions restores infectivity, showing that only a few spikes are necessary for receptor binding and membrane fusion.

Genetika, 1992 Oct, 28(10), 23 - 8
{Preparation and analysis of mutants of rhizospheric pseudomonads, resistant to toxic analogs of tryptophan and phenylalanine}; Mordukhova EA et al.; The absence of plasmids in strains of fluorescent pseudomonads characterized by high level of synthesis of phytohormone indole-3-acetic acid (IAA) as well as invariability of this feature in plasmid and non-plasmid variants of strain BSP8 suggests chromosomal control of IAA synthesis by the rhizosphere bacteria tested . Using toxic analogues of aromatic amino acids -5-fluorine-tryptophan and 5-methyl-tryptophan variants were obtained which synthesized and secreted only anthranilic acid . Mutants with resistance to p-fluorine-phenylalanine and capable of secreting tryptophan and/or phenylalanine were found . Testing of the secreting variants failed to reveal any differences between the levels of IAA biosynthesis in comparison with the wild-type strains.

Biochem Int, 1992 Oct, 28(2), 287 - 95
Enhancing potency of liposomal monensin on ricin cytotoxicity in mouse macrophage tumor cells; Madan S et al.; Monensin, a carboxylic ionophore, which is known to disrupt intracellular trafficking of proteins was intercalated in liposomes and its effect on the stability of liposomes and cytotoxicities of ricin and Pseudomonas exotoxin A in mouse macrophage tumor cells J774A.1 was studied . Stability of liposomes containing monensin was comparable to liposomes without monensin . The cytotoxicity of ricin and Pseudomonas exotoxin A was significantly enhanced by 1nM liposomal monensin (15.7 and 3.6 fold respectively) . The enhancing potency of monensin in neutral and negative vesicles was found to be similar, while it was drastically reduced in positive vesicles . The specific uptake of 125I-gelonin from neutral and negative vesicles was not significantly different, whereas from positive vesicles no uptake was observed . Serum strongly influenced the binding at 4 degrees C of positive vesicles as well as the enhancing potency of monensin in these vesicles . Monensin in neutral and negative vesicles significantly reduced the lag period of ricin action, while in positive vesicles, it had no effect . These studies clearly indicate that liposomes could be used as a delivery vehicle for monensin.

Aust N Z J Surg, 1992 Oct, 62(10), 780 - 4
Splenic abscess; Ooi LL et al.; Isolated splenic abscess is an uncommon condition . Seven cases seen between 1980 and 1990 are reviewed . The clinical presentation is non-specific and diagnosis is usually delayed . Computerized tomography allowed for accurate diagnosis in all cases . Pseudomonas species as a causative organism is reported to be rare, but were present in three of the present cases . Antibiotic therapy alone is insufficient and splenectomy remains the treatment of choice.

J Appl Bacteriol, 1992 Oct, 73(4), 317 - 23
Species of Pseudomonas obtained at 7 degrees C and 30 degrees C during aerobic storage of lamb carcasses; Prieto M et al.; A total of 268 strains of Pseudomonas isolated during storage life of lamb carcasses was identified to species level . One-hundred and thirteen strains obtained at 30 degrees C were Ps . fragi (51), Ps . lundensis (17), Ps . fluorescens biovars I (10), III (9) and VI (1), Ps . putida biovar A (8 strains) and unidentified (17 strains) . Species and biovars isolated at 7 degrees C (155) were Ps . fragi (101), Ps . lundensis (32), Ps . fluorescens biovar I (6), Ps . putida biovar A (8) and unidentified (8) . Numerical analysis (82% SSM, UPGMA) of 'psychrotrophic' and 'mesophilic' strains resulted in the formation of nine and eight clusters respectively . The dendrograms obtained exhibited similar structures . Most of the strains of Ps . lundensis and Ps . fragi clustered together . Strains of this latter species also joined the type strain of Ps . testosteroni and appeared included with phenons containing the Ps . putida strains . There were clusters made up exclusively of strains assigned to one biovar or group (Ps . fluorescens biovars I and II and unidentified) . A high level of similarity was observed between clusters of Ps . fluorescens biovar I and those containing the Ps . fragi-Ps . lundensis complex (> 74% SSM) and Ps . lundensis (> 80%) . The recovery of pseudomonads seemed to be affected by the sampling day.

Biochem Med Metab Biol, 1992 Oct, 48(2), 149 - 58
Biosynthesis and characterization of (S)-and (R)-3-hydroxy-3-methylglutaryl coenzyme A; Bischoff KM et al.; (S)-3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), the physiologic substrate of HMG-CoA reductase and of HMG-CoA lyase, is available commercially only as (R,S)-HMG-CoA, a mixture of diastereomers . To provide (S)-HMG-CoA for our continuing investigation of HMG-CoA reductase, we used homogeneous, overexpressed Pseudomonas mevalonii HMG-CoA reductase (EC 1.1.1.88) and an NAD(+)-regenerating system to convert (R)-mevalonate to (S)-HMG-CoA with an overall yield in excess of 50% . We also used P . mevalonii HMG-CoA lyase (EC 4.1.3.4) to prepare (R)-HMG-CoA from (R,S)-HMG-CoA . Each diastereomer was then isolated by ion-exchange chromatography . Large-scale preparations provide for economical production of (S)-HMG-CoA, particularly when recovered coenzyme A is recycled . (S)-HMG-CoA was evaluated as a substrate, and (R)-HMG-CoA as an inhibitor, for the P . mevalonii enzymes HMG-CoA reductase and HMG-CoA lyase, and for Syrian hamster HMG-CoA reductase (EC 1.1.1.34) . For both HMG-CoA reductases, (R)-HMG-CoA inhibited competitively with respect to (S)-HMG-CoA . The ratio Ki/Km was 0.7 +/- 0.1 and 0.6 +/- 0.2 for the bacterial and hamster enzymes, respectively . By contrast, (R)-HMG-CoA did not inhibit P . mevalonii HMG-CoA lyase.

Biol Chem Hoppe Seyler, 1992 Oct, 373(10), 1001 - 7
Purification and properties of 4-halobenzoate-coenzyme A ligase from Pseudomonas sp . CBS3; Loffler F et al.; The bacterial strain Pseudomonas sp . CBS3 possesses a multi component enzyme system which converts 4-chlorobenzoate to 4-hydroxybenzoate . In the first step 4-chlorobenzoate is activated in a coenzyme A, ATP and Mg(2+)-dependent reaction to 4-chlorobenzoyl-coenzyme A . ATP is cleaved thereby into AMP and pyrophosphate . The involved 4-chlorobenzoate-coenzyme A ligase was purified to apparent homogeneity by a 6-step purification procedure . The native enzyme had an apparent molecular mass of 115000 Da and was composed of two identical polypeptide subunits of 57 kDa . The enzyme displayed an isoelectric point of 5.3 . The maximal initial rate of catalysis was achieved in 100mM Tris/HCl or Tricine/NaOH buffer, pH 8.4, at 35 degrees C . Under these conditions the apparent Km values for ATP, coenzyme A and 4-chlorobenzoate were 2.4 to 3.5 mM, 0.11 to 0.19mM and 0.05 to 0.065mM, respectively . Vmax was 111.6 mumol/(min x mg protein) . The N-terminal amino-acid sequence was determined . 4-Halobenzoates were preferentially converted to the corresponding thioesters . Therefore, the enzyme was named 4-halobenzoate-coenzyme A ligase.

Hum Pathol, 1992 Oct, 23(10), 1172 - 7
Graft eosinophilia in lung transplantation; Yousem SA; Graft eosinophilia was observed in lung biopsies from nine patients who received lung allografts . Five cases were associated with moderate to severe acute cellular rejection and responded well to steroid therapy . In this group the eosinophilia occurred early after transplantation and was associated with an elevated white blood cell count and occasional peripheral blood (one of five cases) and bronchoalveolar lavage (one of five cases) eosinophilia . A second group of four patients had graft eosinophilia due to infectious agents . In these cases patients frequently had underlying bronchiolitis obliterans (two of four cases) and developed tissue eosinophilia late after transplantation . Bronchoalveolar lavage cell profiles often demonstrated dramatic eosinophilia . Histologically, the biopsy specimens displayed an acute eosinophilic pneumonia, which was attributed to Aspergillus sp (two cases), coxsackie A2 virus (one case), and Pseudomonas maltophilia (one case) . Two patients in this group died from infection . While eosinophils are a frequent cellular component of acute rejection reactions, they also may be the dominant cellular component in graft infection.

Cancer Res, 1992 Oct 1, 52(19), 5379 - 85
Monovalent immunotoxin containing truncated form of Pseudomonas exotoxin as potent antitumor agent; Debinski W et al.; Recombinant truncated forms of Pseudomonas exotoxin A that lack the cell binding domain of Pseudomonas exotoxin A were coupled to an F(ab') fragment of a monoclonal antibody HB21 directed against the human transferrin receptor . One of these was NlysPE40 . The other, NlysPE38QQR, has two amino groups on residues near the NH2-terminus and has no amino groups near the COOH-terminus . The proteins were linked by a stable thioether bond that connected the sulfhydryl group present in the hinge region of the antibody fragment to an amino group on the toxin . The F(ab')-PE40 immunotoxin, containing NlysPE40, exhibited potent cytotoxic activity on human carcinoma cell lines with a concentration of immunotoxin at which isotope incorporation falls by 50% when compared to nontreated cells (ID50) of 5.3 pM (0.5 ng/ml) on both the epidermoid carcinoma A431 and on the colon carcinoma Colo205 . Immunotoxins made with whole antibody were considerably less active, with an ID50 of 15.9 pM (3.1 ng/ml) on these cell lines . F(ab')-PE38QQR, the immunotoxin containing NlysPE38QQR, was found to be the most active agent with an ID50 of 1.05 pM (0.1 ng/ml) on A431 cells . The greater cytotoxicity of immunotoxins containing fragmented antibody was probably due to the higher binding affinity of F(ab') conjugates in comparison to whole antibody conjugates to the transferrin receptor . The increase in cytotoxic activity of the immunotoxin made with NlysPE38QQR than that with NlysPE40 may reflect selective coupling of the toxin through NH2-terminal amino groups . The monovalent and divalent immunotoxins had dose-dependent antitumor effects on human epidermoid carcinoma xenografts in nude mice . A431 tumors completely regressed in all animals at a total dose of 105 pmol (10 micrograms) of F(ab')-PE38QQR and of 154 pmol (30 micrograms) of IgG-PE38QQR . Furthermore, the F(ab') immunotoxin was less toxic to mice than the conjugate containing IgG (840 pmol or 80 micrograms of total dose causing measurable adverse effects versus 208 pmol or 40 micrograms, respectively) . Thus, a truncated Pseudomonas exotoxin A molecule coupled to the F(ab') fragment of an antibody is more active and less toxic in mice than an immunotoxin made with a whole antibody . Therefore, the therapeutic index for the monovalent immunotoxin is about four times better than that for the divalent immunotoxin.

J Surg Res, 1992 Oct, 53(4), 357 - 61
Effect of cyclosporine on adrenocortical response to injury and infection; Kapur S et al.; The effects of cyclosporine administration on the adrenocortical response to the severe stress of burn wound sepsis were studied in Wistar rats . Animals were treated with cyclosporine (10 mg/kg/day) or saline by gavage for 10 days, then subjected to 30% scald burns with wound inoculation with Pseudomonas . Animals were sacrificed on Postburn Days (PBDs) 1, 4, and 7 for determination of serum corticosterone and ACTH levels and adrenal weights and histology . Adrenal glands from animals sacrificed on PBD 7 were also analyzed for DNA, RNA, and protein content . Cyclosporine treatment without injury had no significant effect on body weight gain, adrenal mass, or baseline ACTH or corticosterone levels . During sepsis, cyclosporine-treated animals demonstrated a significantly diminished adrenocortical response compared to those given only saline . Serum corticosterone levels in the cyclosporine group were 45, 53, and 62% lower on PBDs 1, 4, and 7, respectively, than in saline-treated controls (P < 0.01 on each day) . ACTH levels were 43 and 36% lower in cyclosporine-treated animals on PBDs 4 and 7, respectively, compared to the saline-treated group (P < 0.05 on each day) . Adrenal hyperplasia occurred in both groups by PBD 7, but increases in adrenal mass and in histologic changes associated with hyperplasia (lipid depletion, vascular dilation) were less pronounced in cyclosporine-treated animals compared to those receiving saline, while adrenal composition remained similar between the two groups . Thus, cyclosporine administration is associated with an attenuated adrenocortical response to the stress of sepsis due to diminished circulating levels of ACTH.

Biosci Biotechnol Biochem, 1992 Oct, 56(10), 1584 - 8
A novel sulfatase from Pseudomonas testosteroni hydrolyzing lithocholic acid sulfate; Tazuke Y et al.; Pseudomonas testosteroni ATCC 11996 was found to produce a novel bile acid sulfate sulfatase that hydrolyzes the sulfate ester bond in lithocholic acid sulfate (LCA-S) . The enzyme synthesis was induced by several kinds of bile acids including LCA-S . Mn2+ functioned as an essential component for the enzyme synthesis and SO4(2-) suppressed it . This sulfatase hydrolyzes LCA-S to isolithocholic acid and sulfuric acid with inversion of alpha- to beta-configuration of the hydroxyl group at the third position of lithocholic acid.

Biosci Biotechnol Biochem, 1992 Oct, 56(10), 1536 - 40
Isolation, purification, and characterization of a new enzyme from Pseudomonas sp . M-27, carboxypeptidase G3; Yasuda N et al.; A new type of carboxypeptidase was found in a strain of Pseudomonas sp . M-27 isolated from soil . The cell-free extract, solubilized by colistin sulfate, was purified to homogeneity . This enzyme had a single peak with a molecular weight of 60,000 on a calibrated Superdex column and consisted of four subunits of identical molecular weights (M(r): 15,000) . The enzyme hydrolyzed predominantly acidic peptides and N-acyl amino acids with Glu or Asp in the C-termini . This enzyme was not strongly affected by thiol enzyme inhibitors (PCMB, iodoacetic acid) or serine protease inhibitors (DFP, PMSF), but was inhibited by metal chelators . The enzyme resembles carboxypeptidase G1 or G2 in its glutamate-releasing activity . However, it acts not only on the L-form but also on the D-form of acidic amino acids and shows affinity for the long-chain fatty acyl group but not the benzoyl group . Thus, as this enzyme differs from carboxypeptidase G1 or G2, it was named carboxypeptidase G3.

J Bacteriol, 1992 Oct, 174(20), 6653 - 8
Characterization of Tn5 mutants deficient in dissimilatory nitrite reduction in Pseudomonas sp . strain G-179, which contains a copper nitrite reductase; Ye RW et al.; Tn5 was used to generate mutants that were deficient in the dissimilatory reduction of nitrite for Pseudomonas sp . strain G-179, which contains a copper nitrite reductase . Three types of mutants were isolated . The first type showed a lack of growth on nitrate, nitrite, and nitrous oxide . The second type grew on nitrate and nitrous oxide but not on nitrite (Nir-) . The two mutants of this type accumulated nitrite, showed no nitrite reductase activity, and had no detectable nitrite reductase protein bands in a Western blot (immunoblot) . Tn5 insertions in these two mutants were clustered in the same region and were within the structural gene for nitrite reductase . The third type of mutant grew on nitrate but not on nitrite or nitrous oxide (N2O) . The mutant of this type accumulated significant amounts of nitrite, NO, and N2O during anaerobic growth on nitrate and showed a slower growth rate than the wild type . Diethyldithiocarbamic acid, which inhibited nitrite reductase activity in the wild type, did not affect NO reductase activity, indicating that nitrite reductase did not participate in NO reduction . NO reductase activity in Nir- mutants was lower than that in the wild type when the strains were grown on nitrate but was the same as that in the wild type when the strains were grown on nitrous oxide . These results suggest that the reduction of NO and N2O was carried out by two distinct processes and that mutations affecting nitrite reduction resulted in reduced NO reductase activity following anaerobic growth with nitrate.

Arch Fr Pediatr, 1992 Oct, 49(8), 721 - 3
{Chronic intestinal pseudo-obstruction and delayed myenteric ganglion cell maturation}; Hugot JP et al.; BACKGROUND . Enteric nerve cells begin to mature during the last trimester of pregnancy and become mature only after birth . The degree of maturation seems to be related to bowel motility . CASE REPORT . A girl was born from a pregnancy complicated by hydramnios, and did not pass meconium before the 64th hr of life . Barium enema on day 4 showed a left microcolon with no distension of the transverse colon . Tests for cystic fibrosis were negative . On day 12, the patient presented with septicemia due to Pseudomonas maltophylia . Parenteral alimentation by central catheter was instituted . Surgical rectal biopsy showed that the number of ganglion cells was normal but the cells were immature . Progressive feeding was possible for the 3rd month of life . A second rectal biopsy at 3 1/2 months showed some remaining immature ganglion cells . CONCLUSION . Immature ganglion cells can account for neonatal functional intestinal obstruction, as has been established for the small left colon syndrome . The progressive loss of symptoms seems to be correlated with histological maturation of the neurological apparatus of the large bowel . Severe complications, such as occlusion, sepsis, nutritional disorders can occur during this long period of functional intestinal obstruction.

Jpn J Med Sci Biol, 1992 Oct-Dec, 45(5-6), 231 - 45
Serosurveillance for double infection with Pseudomonas pseudomallei in tuberculous patients; Kanai K et al.; The serosensitivity to Pseudomonas pseudomallei antigen in pulmonary tuberculous patients was surveyed in Ubon Ratchathani Province, a melioidosis-endemic and tuberculosis-prevalent area in Thailand . Indirect hemagglutination assay (IHA) and indirect immunofluorescent assay (IFA) for IgM and IgG were employed for the measurement of antibody levels, with cut-off points set at 1:160, 1:8, and 1:32, respectively . Retrospective protocol survey of clinical data was also conducted . From these studies, however, no evidence was obtained to show that tuberculous patients have a disposition to acquire double-infected with P . pseudomallei and to develop melioidosis . The serosensitivity was never higher than that of the general population of the province as represented by healthy blood donors, nor related with the clinical severeness . Tuberculosis and melioidosis appear to be mutually independent diseases without showing interrelated prevalence in the endemic area.

Biochim Biophys Acta, 1992 Sep 21, 1110(1), 37 - 44
Monensin intercalation in liposomes: effect on cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in CHO cells; Madan S et al.; Monensin, a carboxylic ionophore was intercalated in liposomes (liposomal monensin) and its effect on cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in CHO cells was studied . Intercalation of monensin in liposomal bilayer is found to have no effect on its stability and interaction with cells . Liposomal monensin (1 nM) substantially enhance the cytotoxicities of ricin (62-fold) and Pseudomonas exotoxin A (11.5-fold) while it has no effect on diphtheria toxin . This observed effect is highly dependent on the liposomal lipid composition . The potentiating ability of monensin (1 nM) in neutral vesicles is significantly higher (2.2-fold) as compared to negatively charges vesicles . This ability is drastically reduced by incorporation of stearylamine in liposomes and is found to be dependent on the density of stearylamine as well as on the concentration of serum in the medium . Monensin in liposomes containing 24 mol% stearylamine has a very marginal effect on the cytotoxicity of ricin (7.5-fold) which is further reduced (1.5-fold) in the presence of 20% serum . The uptake of 125I-gelonin from neutral vesicles is significantly higher (approximately 2.0-fold) than that from the negative vesicles . The uptake from positive vesicles is highly dependent on the concentration of stearylamine . The reduction in the lag period (30 min) of ricin action by monensin in neutral and negative vesicle is comparable with free monensin . However, monensin in positive vesicle has no effect on it . These studies have suggested that liposomes could be used as a delivery vehicle for monensin for selective elimination of tumor cells in combination with hybrid toxins.

FEMS Microbiol Lett, 1992 Sep 15, 75(2-3), 129 - 33
Extraction and characterization of lipopolysaccharide from Pseudomonas pseudomallei; Kawahara K et al.; The best yield of lipopolysaccharide (LPS) of Pseudomonas pseudomallei GIFU 12046 was obtained by extraction of defatted cells by phenol/chloroform/petroleum ether . The LPS showed a smooth character on SDS-polyacrylamide gel electrophoresis and contained D-glucose, L-glycero-D-manno-heptose, and D-glucosamine as the main sugar components, and 3-hydroxypalmitic acid as an amide-linked fatty acid . The growth conditions did not affect the electrophoresis profile and chemical composition of LPS . 2-Keto-3-deoxyoctonic acid was not detectable, and mild acid hydrolysis could not liberate free lipid A, suggesting that the linkage between inner core and lipid A was stable against acid hydrolysis, and the structure of this region is similar to that of P . cepacia, which has close taxonomic relationship with P . pseudomallei.

Cancer Res, 1992 Sep 15, 52(18), 4995 - 5001
Acidic fibroblast growth factor-Pseudomonas exotoxin chimeric protein elicits antiangiogenic effects on endothelial cells; Merwin JR et al.; It has recently been shown that chimeric toxins composed of acidic fibroblast growth factor fused to mutant forms of Pseudomonas exotoxin (aFGF-PE) are cytotoxic to a variety of tumor cell lines with FGF receptors . Although aFGF-PE might be considered as a possible chemotherapeutic toxin, limited knowledge is available concerning its effect on endothelia . This study investigates whether one of the aFGF-PE fusion proteins, aFGF-PE664GluKDEL, can function as an anti-angiogenic agent . Protein synthesis studies using rat epididymal fat pad microvascular endothelial cells (RFCs) indicated that after 24 h in culture, aFGF-PE had a significant inhibitory effect on protein synthesis at concentrations greater than 100 ng/ml . In cultures incubated with 1000 ng/ml aFGF-PE, RFC protein synthesis was inhibited as much as 83% . RFCs were also cultured in a 3-dimensional type I collagen gel and incubated with either transforming growth factor beta 1, aFGF-PE, or a combination of both . Transforming growth factor beta 1 elicits in vitro angiogenesis in these 3-dimensional cultures which consist of rapid formation of complex tubular structures . Transforming growth factor beta 1-treated RFCs incubated with aFGF-PE were unable to produce this angiogenic response, nor were they able to migrate out of the 3-dimensional culture to form a monolayer as shown by controls . Cell viability analyses showed that aFGF-PE produced a dose-dependent toxic effect which ranged from 10 to 90% cell death . Competition assays in which the chimeric toxin was preincubated with antibodies to aFGF resulted in an 89% reversal of the inhibitory effects of aFGF-PE on endothelial cells . Acidic FGF-PE with a mutation in the ADP ribosylation domain of PE was inactive in both 2-dimensional and 3-dimensional cultures . These data show that aFGF-PE has specific in vitro cytotoxic, antiangiogenic, and antimigratory effects on microvascular endothelia.

FEMS Microbiol Lett, 1992 Sep 15, 75(2-3), 111 - 4
Use of long-chain fatty acid-CoA ligase (AMP-forming) from Pseudomonas fragi for the 'in vitro' synthesis of natural penicillins; Fernandez-Valverde M et al.; Five different naturally occurring penicillins containing as side chains hexanoic, trans-3-hexenoic, heptanoic, octanoic or trans-3-octenoic acids have been synthesized 'in vitro' by coupling long-chain fatty acid-CoA ligase (AMP-forming) (EC 6.2.1.3) from Pseudomonas fragi (LFCoA-L) with acyl-CoA: 6-aminopenicillanic acid acyltransferase (AT) from Penicillium chrysogenum . The quantity of penicillin produced was directly related with the carbon length of the side chain precursor tested, being maximal with octanoic acid . Fatty acids with a lower length than C5 were not recognized as substrates and nor were certain aromatic molecules.

Med J Aust, 1992 Sep 7, 157(5), 332 - 4
A case of human melioidosis originating in south-west Western Australia; Golledge CL et al.; OBJECTIVE: To report the first human case of autochthonous melioidosis in temperate Australia (latitude 31 degrees 10'S) and to describe the extent of the presence of the causative agent, Pseudomonas pseudomallei, in southwest Western Australia . CLINICAL FEATURES: A 45-year-old man living on a hobby farm was admitted to hospital for investigation of lung lesions, weight loss and low grade fevers . P . pseudomallei was cultured from material from an aspiration biopsy of a mediastinal mass . INTERVENTION AND OUTCOME: The patient was successfully treated with a regimen of ceftazidime and trimethoprim-sulfamethoxazole . A review of epidemiological data showed that, since 1967, P . pseudomallei has been cultured from animals and soil in a region 50-250 km north-west of Perth, Western Australia, and that pockets of endemicity are found in the districts of Toodyay (where our patient's farm was), Chittering, Ballidu, Gidgegannup, Badgingarra, and Wongan Hills . CONCLUSION: The persistence of the bacterium in animals and soil in south-west Western Australia demonstrates abundantly that P . pseudomallei can exist opportunely beyond its traditional tropical habitat . It is likely that there will be further clinical cases of melioidosis originating in this region, and that the boundaries of endemicity in Australia will expand further.

Cell Immunol, 1992 Sep, 143(2), 324 - 34
In vitro and in vivo suppression of interleukin-2-activated killer cell activity by chimeric proteins between interleukin-2 and Pseudomonas exotoxin; Puri RK et al.; The biological effects of IL-2 are mediated through high (complex of alpha and beta chain) or intermediate (beta chain) affinity IL-2 receptors . Previously, chimeric proteins composed of IL-2 and Pseudomonas exotoxin (IL-2-PE) were shown to be specifically cytotoxic to cells bearing IL-2 receptors . It has also been shown that IL-2-PE chimeric proteins can abrogate T cell-mediated immune response in vitro . In the current study, we have investigated the effects of IL-2-PE on LAK activity both in vivo and in vitro . We administered either IL-2-PE40 (comprised of IL-2 and 40-kDa portion of PE) or IL-2-PE66 (comprised of IL-2 and 66-kDa molecule of PE) to normal C57BL/6 mice for 3 or 8 days and LAK activity was assessed in various organs of mice . We found that IL-2-PE40 generated LAK activity in various compartments of mice and the level of activity was slightly lower than that observed with an equivalent amount of recombinant (r) IL-2 alone . However, IL-2-PE66 failed to generate LAK activity which would have been induced due to an equivalent concentration of rIL-2 . IL-2-PE66 also did not induce LAK activity from the splenocytes during in vitro culture while IL-2-PE40 generated good LAK activity . An equivalent amount of IL-2 also generated potent LAK activity . The suppression of LAK activity by IL-2-PE66 was also evident in cells preactivated with IL-2; however, this inhibition was partial . The suppressive activity of IL-2-PE66 was shown to be mediated through IL-2 receptor interactions as excess amounts of rIL-2 were able to abrogate its effect . Both IL-2 toxins were equivalently cytotoxic to IL-2 receptor-bearing HUT 102 cells and both were able to compete from high and intermediate affinity IL-2 receptors . Taken together, our data indicate that IL-2-PE66 is highly cytotoxic to LAK cells while IL-2-PE40 is less cytotoxic . Thus, data from our study and from other published reports indicate that IL-2-PE66 is more potent immunosuppressive agent than IL-2-PE40.

Appl Environ Microbiol, 1992 Sep, 58(9), 2879 - 85
Characterization of a novel Pseudomonas sp . that mineralizes high concentrations of pentachlorophenol; Radehaus PM et al.; A pentachlorophenol (PCP)-mineralizing bacterium was isolated from polluted soil and identified as Pseudomonas sp . strain RA2 . In batch cultures, Pseudomonas sp . strain RA2 used PCP as its sole source of carbon and energy and was capable of completely degrading this compound as indicated by radiotracer studies, stoichiometric release of chloride, and biomass formation . Pseudomonas sp . strain RA2 was able to mineralize a higher concentration of PCP (160 mg liter-1) than any previously reported PCP-degrading pseudomonad . At a PCP concentration of 200 mg liter-1, cell growth was completely inhibited and PCP was not degraded, although an active population of Pseudomonas sp . RA2 was still present in these cultures after 2 weeks . The inhibitory effect of PCP was partially attributable to its effect on the growth rate of Pseudomonas sp . strain RA2 . The highest specific growth rate (mu = 0.09 h-1) was reached at a PCP concentration of 40 mg liter-1 but decreased at higher or lower PCP concentrations, with the lowest mu (0.05 h-1) occurring at 150 mg liter-1 . Despite this reduction in growth rate, total biomass production was proportional to PCP concentration at all PCP concentrations degraded by Pseudomonas sp . RA2 . In contrast, final cell density was reduced to below expected values at PCP concentrations greater than 100 mg liter-1 . These results indicate that, in addition to its effect as an uncoupler of oxidative phosphorylation, PCP may also inhibit cell division in Pseudomonas sp . strain RA2.(ABSTRACT TRUNCATED AT 250 WORDS)

Anal Biochem, 1992 Sep, 205(2), 359 - 64
A comparison of substrates for quantifying the signal from a nonradiolabeled DNA probe; Kerkhof L; A method for measuring the amount of a nonradiolabeled DNA probe using four detection substrates is described . In preliminary experiments, digoxygenin-labeled DNA was bound to neutral, nylon membranes and detected with anti-digoxygenin antibodies conjugated to alkaline phosphatase . Four substrates {4-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, AttoPhos, and adamantyl 1, 2-dioxetane phosphate (AMPPD)} were assessed for use in a quantitative hybridization assay . Only AttoPhos and AMPPD were found to have detection limits in the low picogram range and to respond linearly to DNA concentrations ranging from 0 to 1250 pg . In subsequent experiments, a 200-bp DNA probe cloned from the marine bacterium Pseudomonas perfectomarina 23S rRNA gene was hybridized to P . perfectomarina genomic DNA and total RNA . The amount of hybridized probe was determined using AttoPhos . Finally, a digoxygenin-labeled oligonucleotide was probed against genomic DNA . Linearity with respect to DNA concentration was observed using both the 200-bp fragment and the oligonucleotide as probes with a final target detection limit of 166 fg . This study demonstrates the substrate AttoPhos can be used to quantify the amount of nonradiolabeled probe hybridized to target with sufficient sensitivity for very dilute samples, such as environmental samples.

Cornea, 1992 Sep, 11(5), 398 - 403
Anti-inflammatory therapy and outcome in a guinea pig model of Pseudomonas keratitis; Ohadi C et al.; Corneal scarring as a consequence of bacterial keratitis is an important cause of visual loss and a major indication for penetrating keratoplasty . Anti-inflammatory agents might be useful in this condition for limiting corneal damage, but benefit from adjunctive anti-inflammatory therapy has never been demonstrated . In this limited pilot study, we compared the effect on clinical outcome of treating Pseudomonas keratitis in guinea pigs with prednisolone (a corticosteroid), flurbiprofen (a cyclo-oxygenase inhibitor), nordihydroguaiaretic acid (a lipoxygenase inhibitor), and a leukotriene antagonist, SKF104353 {R-(R*, S*)}-beta-{(2-carboxyethyl) thio-alpha-hydroxy-2-(8-phenyloctyl) benzenepropanoic acid, zinc salt} . None of the anti-inflammatory agents prevented sterilization of ulcers with antibiotic (ofloxacin) therapy . Therapy with the leukotriene antagonist appeared to reduce infiltrate size more quickly and produce a more rapid reduction in lesion size, but the differences were not statistically significant . Sample size calculations suggest that very large numbers of animals would be required to prove efficacy . The role of anti-inflammatory agents in reducing the stromal destruction caused by bacterial keratitis remains controversial.

Ir Med J, 1992 Sep, 85(3), 110 - 1
Home intravenous therapy using a silastic long line catheter in cystic fibrosis patients; Pradeepkumar VK et al.; Young patients with cystic fibrosis are now accustomed to having regular periods of intravenous therapy to help in the management of their recurring respiratory infections particularly due to pseudomonas organisms . Most patients will eventually experience difficulties in accepting recurring intravenous cannulations and the life span of the conventional intravenous cannula is frequently brief . We have compared percutaneous long line silastic catheters with conventional intravenous cannulas as intravenous access in children with cystic fibrosis being treated for pulmonary exacerbations . We conclude that silastic long lines are superior to conventional cannulas in terms of patient tolerance, reduction in the number of hospital admissions, reduction in the number of repeat venepunctures/cannulations and reduction in local complications . The high degree of patient acceptability has been impressive and has increased with the enthusiasm of children with cystic fibrosis for more frequent intravenous therapy.

J Infect, 1992 Sep, 25(2), 139 - 46
Homogeneity of lipopolysaccharide antigens in Pseudomonas pseudomallei; Pitt TL et al.; An antiserum raised against a single strain of Pseudomonas pseudomallei reacted equally in a whole cell agglutination test, an indirect haemagglutination (IHA) test and an ELISA with a panel of 12 strains of the species which had been isolated from human beings and animals in various parts of the Far East and Australia between 1923 and 1990 . Absorption of the serum with either of two strains removed all reactivity of the serum with other strains . Phenol-water extracted lipopolysaccharide (LPS) from a single strain blocked the reactivity of the serum with red cells sensitised with crude extracts of any of the panel of strains, thereby suggesting that the 'common' antigen was LPS . This antigen was not detected in other Pseudomonas species with the exception of Pseudomonas mallei . Protease K-digested extracts of the 12 strains gave highly similar silver-stained LPS banding patterns in gel electrophoresis . Furthermore, immunoblots of LPS with either rabbit or a patient's serum showed identical ladder profiles for each strain . The results suggest that the LPS antigen is highly conserved throughout P . pseudomallei and that this antigen is detected by the IHA test.

Zhonghua Yu Fang Yi Xue Za Zhi, 1992 Sep, 26(5), 287 - 90
{Study of toxication of toxoflavin from Pseudomonas cocovenenans to immunocyte and detoxication}; Yue QA; In order to study the toxication of toxoflavin produced by P . cocovenenans to immunocytes and detoxication, the effects of toxoflavin on rabbit blood culture in the presence of PHA and detoxication function of seven drugs towards this system have been observed by 3H-TdR incorporation assay . The more toxoflavin used, the more reduction of cpm value . The results indicated that toxoflavin in 0.5 microgram/ml or more suppressed the growth of cells to even still lower than that of control in which PHA was absent; and when 0.1 microgram/ml of toxoflavin were used, the cpm value began to rise again . The effects of toxoflavin on lymphocyte and other blood cells were studied under the microscope and the naked-eye . The detoxication effects of the seven drugs on toxoflavin were observed and shown that SOD, GSH, Cyt . C and VC were most effective in protecting the lymphocytes from poisoning.

Pediatr Infect Dis J, 1992 Sep, 11(9), 722 - 6
Association of Epstein-Barr virus infection and pulmonary exacerbations in patients with cystic fibrosis; Winnie GB et al.; We observed severe pulmonary exacerbations during primary Epstein-Barr virus (EBV) infection in adolescent patients with cystic fibrosis . Since EBV is not a known respiratory tract pathogen in cystic fibrosis, we studied retrospectively all EBV-susceptible patients ages 6 to 18 years with chronic Pseudomonas respiratory tract colonization hospitalized for a pulmonary exacerbation during an 18-month period . Patients with serologic evidence of primary EBV infection (n = 5) were compared to control patients without EBV (n = 7) . Before admission the groups had similar pulmonary function tests, clinical scores and frequency of hospitalization . On admission patients with EBV had significant weight loss, lower pulmonary function tests and lower clinical scores compared with controls . All remained significantly different 6 months after admission . Frequency of exacerbations requiring hospitalization increased after EBV infection but remained unchanged in controls . Primary EBV infection can be associated with severe pulmonary exacerbations and subsequent deterioration in clinical course in cystic fibrosis patients.

Anal Biochem, 1992 Sep, 205(2), 263 - 70
A method for increasing the yield of properly folded recombinant fusion proteins: single-chain immunotoxins from renaturation of bacterial inclusion bodies; Buchner J et al.; Many proteins produced in Escherichia coli accumulate in inclusion bodies . We have systematically evaluated the parameters that affect the refolding and renaturation of enzymatically active molecules from bacterial inclusion bodies containing a recombinant single-chain immunotoxin, B3(Fv)-PE38KDEL . This recombinant molecule is composed of the variable domains of monoclonal antibody B3 (B3(Fv)) fused to a truncated mutant form of Pseudomonas exotoxin A (PE38KDEL) . This immunotoxin kills carcinoma cells in vitro, causes tumor regression in animal tumor models, and is being developed as an anti-cancer therapeutic agent (Brinkmann et al., 1991, Proc . Natl . Acad . Sci . USA 88, 8616-8620) . Like many other recombinant proteins, B3(Fv)-PE38KDEL is produced in E . coli in inclusion bodies and must be denatured and refolded to become active . This requires correct folding, formation of native disulfide bonds, and the association of different domains . All these steps are strongly dependent on the renaturation conditions used . Optimum conditions of refolding were obtained by the addition of reduced and oxidized thiol reagents to promote disulfide bond formation and the addition of a labilizing agent such as L-arginine . Furthermore, the necessity to reactivate proteins at low protein concentrations due to its tendency to aggregate at high concentrations was overcome by a step-by-step addition of denatured and reduced protein into the refolding solution . This approach should be useful for the production of active forms of other recombinant proteins.

Circ Res, 1992 Sep, 71(3), 640 - 5
In vitro effects of a recombinant toxin targeted to the fibroblast growth factor receptor on rat vascular smooth muscle and endothelial cells; Biro S et al.; The dominant mechanism responsible for restenosis after angioplasty is believed to be the activation of medial smooth muscle cells (SMCs), leading to their proliferation, migration to the subintima, and further proliferation . To develop novel strategies that might inhibit or prevent restenosis, we previously used a chimeric toxin composed of transforming growth factor-alpha (which targets the epidermal growth factor receptor) and mutated Pseudomonas exotoxin to preferentially recognize and kill rapidly proliferating, versus quiescent, vascular SMCs . We have recently cloned and expressed a recombinant gene encoding Pseudomonas exotoxin with a mutated (nonfunctional) cell recognition domain fused with the ligand acidic fibroblast growth factor, termed aFGF-PE66(4Glu)KDEL; thus, this recombinant toxin targets the fibroblast growth factor receptor . In the present study, we evaluated the relative effects of this chimeric toxin on quiescent versus rapidly proliferating vascular SMCs and also determined whether aFGF-PE66(4Glu)KDEL exerted different effects on SMCs versus endothelial cells . Rapidly proliferating SMCs (grown in 10% fetal bovine serum) were very sensitive to the cytotoxic effects of aFGF-PE66(4Glu)KDEL, whereas cytotoxicity was significantly less when the SMCs were in a quiescent state (grown in medium supplemented with 0.5% fetal bovine serum) . The chimeric toxin was also significantly less cytotoxic against endothelial cells . Competition studies using excess acidic fibroblast growth factor indicated that the cytotoxic effects are specifically mediated by the fibroblast growth factor receptor . Thus, the present studies suggest a potentially expanded role of recombinant toxin therapy in restenosis: multiple receptors can be targeted, and cytotoxic effects, at least in vitro, can be preferentially directed to rapidly proliferating vascular SMCs, with relative sparing of vascular endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Hua Xi Yi Ke Da Xue Xue Bao, 1992 Sep, 23(4), 377 - 80
{Studies on human chorionic gonadotropin (HCG)-binding protein from Pseudomonas maltophilia by ligand blotting assay}; Li X et al.; A ligand blotting technique was developed to study the HCG-binding protein from Pseudomonas maltophilia after size separation by SDS-polyacrylamide gel electrophoresis under nonreducing conditions . The separated proteins were transferred to a nitrocellulose sheet, which was subsequently incubated with {125I}iodo HCG, and subjected to determination for radioactivity in gamma-counter . A radioactivity peak equivalent to an M(r) 70000 appeared, which was not observed when the hormone incubation was performed in the presence of an excess of unlabeled HCG . The peak also disappeared when the protein samples were treated with reducing agent, which showed that integrity disulfide bonds of the protein was essential for the protein-hormone interaction . In addition, position of the radioactivity peak which was due to the binding of {125I}iodo HCG to western blots of the HCG-binding protein was corresponding to that of the antibodies against the HCG-binding protein recognizing a 70000 protein on the western blots . These results show that the HCG-binding protein from Pseudomonas maltophilia is an M(r) 70000 protein and that the HCG-binding protein contains at least one disulfide bond essential to its binding activity.

J Med Assoc Thai, 1992 Sep, 75(9), 536 - 41
Cerebral abscesses due to Pseudomonas pseudomallei; Kasantikul V et al.; Two cases of cerebral abscesses caused by Pseudomonas pseudomallei are reported . The first case, a 51-year-old women had a sudden onset of progressive right hemiparesis and right facial palsy and died within 7 days . Postmortem examination disclosed brain abscess in association with disseminated infection outside the central nervous system . The second case, a 9-year-old boy displayed cerebral abscesses as an isolated manifestation . Recovery occurred after treatment with ceftazidime . Review of the ten case reports of cerebral melioidosis revealed that the lesion occurred in patients of all ages and was more common in men than in women . The frontoparietal lobe was the most common location . Fever, headache, and hemiparesis were frequent clinical manifestations while seizures, ataxia were uncommon . CT scanning, serum antibody titer along with hemoculture were useful investigate tools . The importance of early diagnosis and prompt treatment is emphasized for this fatal but treatable disease.

Hua Xi Yi Ke Da Xue Xue Bao, 1992 Sep, 23(3), 301 - 4
{Comparative studies on LH/CG receptor from rat testes and hCG-binding protein from Pseudomonas maltophilia by antibodies against the hCG-binding protein}; Li X et al.; An indirect immunoprecipitation experiment was developed to characterize the antigenic determinants of the hCG-binding protein and the rat testicular LH/CG receptor . The hCG-binding protein preincubated with 125I iodo hCG was immunologically precipitated by the antibodies against the hCG-binding protein in the presence of goat anti-rabbit IgG . Similarly, the LH/CG receptor solubilized with 1% triton X-100 from the rat testes was also immunoprecipitated by the antibodies . Furthermore, the antibodies did not inhibit the binding of 125I iodo hCG to the hCG-binding protein or to the solubilized rat testicular LH/CG receptor . From these results it can be concluded that there exists the sharing of antigenic determinants between the hCG-binding protein from Pseudomonas maltophilia and the LH/CG receptor from the rat testes and that the antibodies specifically recognize the antigenic determinants outside the hCG-binding sites on the rat testicular LH/CG receptor . Therefore, the antibodies against the hCG-binding protein are of potential applications to studies on structure and function of the mammalian LH/CG receptors.

Chin Med J (Engl), 1992 Sep, 105(9), 775 - 9
Pseudomonas pseudomallei and melioidosis in China; Li L et al.; Though melioidosis has been reported since 1912, it is still a new problem for most Chinese physicians and medical scientists . Because of the lack of understanding and clinical experiences in melioidosis, none had been reported until 1990 in the mainland of China . In view of China's open policy and health work the natural foci of Pseudomonas pseudomallei in South China, ie . Hainan, Guangdong and Guangxi Provinces, should be given sufficient concern . Efforts should be made in training physicians in the endemic areas to improve their knowledge about the disease and to avoid misdiagnosis.

J Biol Chem, 1992 Aug 25, 267(24), 16872 - 7
A recombinant form of Pseudomonas exotoxin directed at the epidermal growth factor receptor that is cytotoxic without requiring proteolytic processing; Theuer CP et al.; Pseudomonas exotoxin A is composed of three structural domains that mediate cell recognition (I), membrane translocation (II), and ADP-ribosylation (III) . Within the cell, the toxin is cleaved within domain II to produce a 37-kDa carboxyl-terminal fragment, containing amino acids 280-613, which is translocated to the cytosol and causes cell death . In this study, we constructed a mutant protein (PE37), composed of amino acids 280-613 of Pseudomonas exotoxin A, which does not require proteolysis to translocate . PE37 was targeted specifically to cells with epidermal growth factor receptors by inserting transforming growth factor-alpha (TGF-alpha) after amino acid 607 near the carboxyl terminus of Pseudomonas exotoxin A . PE37/TGF-alpha was very cytotoxic to cells with epidermal growth factor receptors . It was severalfold more cytotoxic than a derivative of full-length Pseudomonas exotoxin A containing TGF-alpha in the same position, probably because the latter requires intracellular proteolytic processing to exhibit its cytotoxicity, and proteolytic processing is not 100% efficient . Deletion of 2, 4, or 7 amino acids from the amino terminus of PE37/TGF-alpha greatly diminished cytotoxic activity, indicating the need for a proper amino-terminal sequence . In addition, a mutant containing an internal deletion of amino acids 314-380 was minimally active, indicating that other regions of domain II are also required for the cytotoxic activity of Pseudomonas exotoxin A.

J Infect Dis, 1992 Aug, 166(2), 344 - 9
Affinity constants of naturally acquired and vaccine-induced anti-Pseudomonas aeruginosa antibodies in healthy adults and cystic fibrosis patients; Bruderer U et al.; Naturally acquired anti-Pseudomonas aeruginosa antibody fails to afford protection against repeated P . aeruginosa bronchopulmonary exacerbations in cystic fibrosis (CF) patients . In an effort to explain this phenomenon, the titer and affinity constants of serum anti-lipopolysaccharide (LPS) IgG were determined in five study groups: healthy adults before and after immunization with a polyvalent LPS-based vaccine, healthy noncolonized CF patients before and after immunization, nonimmunized CF patients with significantly elevated anti-LPS antibody titers without documented colonization, recently colonized CF patients before and after immunization, and nonimmunized CF patients chronically colonized with P . aeruginosa . Immunization elicited a significant rise in total anti-LPS immunoglobulin levels and affinity constants in both healthy adults and CF patients . Although chronically colonized patients had elevated levels of total anti-LPS antibody, these antibodies possessed affinities at least 100-fold less than those of vaccine-induced antibodies.

J Clin Invest, 1992 Aug, 90(2), 405 - 11
Monoclonal antibody C242-Pseudomonas exotoxin A . A specific and potent immunotoxin with antitumor activity on a human colon cancer xenograft in nude mice; Debinski W et al.; Two immunotoxins were constructed by chemically coupling the monoclonal antibody C242 to Pseudomonas exotoxin A (PE) or a modified form, NlysPE40, that lacks the cell binding domain of PE . Monoclonal antibody C242 recognizes a specific sialylated carbohydrate epitope on a high molecular weight membrane glycoprotein present on cells of human colon, pancreatic, and cervical cancers . C242-PE and C242-NlysPE40 were very cytotoxic for cells expressing this antigen with 50% inhibition of protein synthesis occurring on Colo205 cells at 0.2 ng/ml (0.9 pM) for C242-PE and 6.0 ng/ml (31 pM) for C242-NlysPE40 . The two immunotoxins also exhibited a strong antitumor effect on a human colon cancer xenograft grown in nude mice . The specificity and potency of these two C242 immunotoxins warrant their further development for the treatment of cancer.

Burns, 1992 Aug, 18(4), 321 - 5
Microskin autograft with pigskin xenograft overlay: a preliminary report of studies on patients; Lin SD et al.; Split-thickness pigskin graft (STPSG) was used to replace allograft skin for microskin grafting in 16 patients, nine of whom were burn patients, five suffered from traumatic defects and two from diabetic ulcers . The expansion ratios used in these patients ranged from 8:1 to 12:1 . The STPSG preparation described was found to be safe for clinical application . The autogenous donor skin was excised from the inguinal area, and the donor site was primarily closed . There were no instances of donor site morbidity . The majority of the STPSG overlays adhered to the wound firmly . Histological examination showed that the microskin grafts proliferated actively immediately beneath the STPSG overlay . The time for the wound to be fully resurfaced varied from 13 to 21 days depending on the expansion ratio employed . There were only two episodes of pseudomonas infection and no further grafting was required in any of the patients . In this study the pigskin xenograft was found to provide a suitable environment for the epithelialization of microskin autografts . When allograft is not available, this is an alternative way of ensuring successful microskin grafting.

J Antibiot (Tokyo), 1992 Aug, 45(8), 1216 - 21
Acaterin, a novel inhibitor of acyl-CoA: cholesterol acyltransferase produced by Pseudomonas sp . A92; Naganuma S et al.; Acaterin, a novel inhibitor of acyl-CoA: cholesterol acyltransferase (ACAT), was isolated from a culture broth of Pseudomonas sp . A92 by Diaion HP-20 column chromatography, solvent extraction and reverse phase HPLC . Spectroscopic analyses of the compound yielded 3-(1-hydroxyoctyl)-5-methyl-2(5H)-furanone as the proposed structure . In the presence of oxidized low density lipoprotein, acaterin inhibited the synthesis of cholesteryl ester in macrophage J774 by 50% at a concentration of 45 microM . Acaterin also inhibited ACAT activity in the rat liver microsomes by 50% at a concentration of 120 microM . Kinetic studies suggested that inhibition of ACAT by acaterin was noncompetitive with respect to oleoyl-CoA.

Appl Environ Microbiol, 1992 Aug, 58(8), 2501 - 4
Utilization of 3-chloro-2-methylbenzoic acid by Pseudomonas cepacia MB2 through the meta fission pathway; Higson FK et al.; Pseudomonas cepacia MB2 grew on 3-chloro-2-methylbenzoate as a sole carbon source by metabolism through the meta fission pathway with the subsequent liberation of chloride . meta pyrocatechase activity in cell extracts was induced strongly by 3-chloro-2-methylbenzoate, but not by nongrowth analogs 4- or 5-chloro-2-methylbenzoate . Although rapid turnover of metabolites precluded direct identification, a mutant strain MB2-G5 lacking meta pyrocatechase activity produced 4-chloro-3-methylcatechol when incubated with 3-chloro-2-methylbenzoate . The catecholic product, confirmed by nuclear magnetic resonance and mass spectral analyses, produced a transient meta fission product (lambda max = 391 nm) from cell extracts of the wild-type MB2 strain . Further confirmation of meta pyrocatechase activity was noted by conversion of 4-chlorocatechol to 2-hydroxy-5-chloromuconic semialdehyde, which was not further metabolized . In contrast to 3-chlorocatechol, which was not metabolized and is known to generate suicidal products, 4-chlorocatechols do not generate acyl halides . Thus, further metabolism of the ring fission products is governed in strain MB2 by their suitability as substrates for the hydrolase.

J Clin Microbiol, 1992 Aug, 30(8), 2084 - 7
Molecular epidemiology of Pseudomonas cepacia determined by polymerase chain reaction ribotyping; Kostman JR et al.; Traditional ribotyping detects genomic restriction fragment length polymorphisms by probing chromosomal DNA with rRNA . Although it is a powerful method for determining the molecular epidemiology of bacterial pathogens, technical difficulties limit its application . As an alternative, polymorphisms were sought in the 16S-23S spacer regions of bacterial rRNA genes by use of the polymerase chain reaction (PCR) . Chromosomal DNA from isolates of Pseudomonas cepacia was used as a template in the PCR with oligonucleotide primers complementary to highly conserved sequences flanking the spacer regions of the rRNA genes . Length polymorphisms in the amplified DNA distinguished unrelated isolates of P . cepacia . Isolates of P . cepacia previously implicated in person-to-person transmission were shown to have identical amplification patterns . These data demonstrate the utility of this new PCR ribotyping method for determining the molecular epidemiology of bacterial species.

Am J Hum Genet, 1992 Aug, 51(2), 245 - 50
Cystic fibrosis patients bearing both the common missense mutation Gly----Asp at codon 551 and the delta F508 mutation are clinically indistinguishable from delta F508 homozygotes, except for decreased risk of meconium ileus; Hamosh A et al.; The glycine-to-aspartic acid missense mutation at codon 551 (G551D), which is within the first nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator (CFTR), is the third most common cystic fibrosis (CF) mutation, with a worldwide frequency of 3.1% among CF chromosomes . Regions with a high frequency correspond to areas with large populations of Celtic descent . To determine whether G551D confers a different phenotype than does delta F508, the most common CF mutation, we studied 79 compound heterozygotes for G551D/delta F508, from nine centers in Europe and North America . Each subject was matched, by age and sex, with a delta F508 homozygote from the same center . A retrospective cohort analysis was performed on the following outcome parameters: age at diagnosis, sweat chloride, meconium ileus at birth, height, weight, weight for height, FVC, FEV1, chest X-ray score, pseudomonas colonization, pancreatic sufficiency, and Shwachman clinical score . There was less meconium ileus among the G551D/delta F508 compound heterozygotes (relative risk 0.33; 95% confidence interval .13-.86), as well as a trend toward later age at diagnosis of pancreatic insufficiency . No statistically significant difference was found between the groups for any other parameter . These results suggest that the CF genotype can be a predictor of pancreatic and intestinal phenotype . Prenatal counseling for the two genotype groups should differ only with respect to probability of meconium ileus . Clinical outcome (after survival of meconium ileus) for G551D/delta F508 compound heterozygotes and delta F508 homozygotes is indistinguishable; therefore, prognostic counseling should not differ.

Biotechnology (N Y), 1992 Aug, 10(8), 905 - 9
Expression of a bacterial phaseolotoxin-resistant ornithyl transcarbamylase in transgenic tobacco confers resistance to Pseudomonas syringae pv . phaseolicola; de la Fuente-Martinez JM et al.; Toxins have been shown to be an important virulence component for most pathovars of Pseudomonas syringae . Here we have examined the role of phaseolotoxin in the virulence mechanism of P . syringae pv . phaseolicola by producing transgenic tobacco plants that express a pathogen-derived toxin-resistant target enzyme . Such plants are insensitive to the toxin and less prone to infection by the pathogen.

Am J Respir Cell Mol Biol, 1992 Aug, 7(2), 214 - 21
Induction of ICAM-1 expression on human airway epithelial cells by inflammatory cytokines: effects on neutrophil-epithelial cell adhesion; Tosi MF et al.; Inflammation of the human airways in diseases such as chronic bronchitis, cystic fibrosis with Pseudomonas endobronchial infection, and possibly asthma during late-phase reactions involves a local influx of neutrophils (PMN) that may participate in airway epithelial injury . PMN-mediated cellular injury is most efficient under conditions of PMN-target cell adhesion . PMN express adhesive glycoproteins of the CD11/CD18 family that are counter-receptors for intercellular adhesion molecule-1 (ICAM-1), found on various cell types . We proposed that adherence by PMN to human airway epithelial cells via ICAM-1 might be an important mechanism in inflammatory airway diseases . We found that although PMN adhere poorly (less than 5%) to monolayers of human tracheal epithelial cells (TEC) in primary culture, they adhere readily (45 to 50%) to an SV40-immortalized line of human TEC, designated 9HTEo- . We also found 6-fold greater surface expression of ICAM-1 on 9HTEo- compared with primary TEC . Blocking surface ICAM-1 on 9HTEo- cells with specific monoclonal antibody inhibited PMN adherence by about 50% . Thus, ICAM-1 plays a major role in this adherence, although it is possible that other epithelial ligands contribute also . Antibodies to CD11a, CD11b, and CD18 on PMN also inhibited PMN-epithelial adherence . Treatment of primary TEC monolayers with the proinflammatory cytokines interleukin-1 (IL-1) or tumor necrosis factor-alpha (TNF-alpha) caused a 3- to 4-fold increase in both cell surface ICAM-1 expression and support of PMN adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)

J Laryngol Otol, 1992 Aug, 106(8), 739 - 40
Pseudomonal supraglottitis occurring in a patient with profound neutropenia secondary to virus-associated haemophagocytic syndrome; Connolly AA et al.; We present a case of virus-associated haemophagocytic syndrome following Epstein-Barr virus infection in which a fulminant pseudomonal supraglottitis developed . Increasingly, unusual pathogens have been found in immunocompromised patients . This is the first reported case of pseudomonal supraglottitis.

J Bacteriol, 1992 Aug, 174(16), 5340 - 5
NAD(P)H-dependent chromium (VI) reductase of Pseudomonas ambigua G-1: a Cr(V) intermediate is formed during the reduction of Cr(VI) to Cr(III); Suzuki T et al.; An NAD(P)H-dependent Cr(VI) reductase (molecular weight = 65,000) was purified from a Cr(VI)-resistant bacterium, Pseudomonas ambigua G-1 . Stoichiometric analysis of the enzymatic reaction showed that the enzyme catalyzed the reduction of 1 mol of Cr(VI) to Cr(III) while consuming 3 mol of NADH as an electron donor . Chromium(VI) was reduced to Cr(V) by one equivalent NADH molecule in the absence of the enzyme . Electron spin resonance analysis showed that Cr(V) species (g = 1.979) was formed during the enzymatic reduction . The amount of Cr(V) species formed was about 10 times larger than that of the nonezymatic reduction . These findings show that the Cr(VI) reductase reduced Cr(VI) to Cr(III) with at least two reaction steps via Cr(V) as an intermediate.

Clin Exp Immunol, 1992 Aug, 89(2), 321 - 4
Decreased polymorphonuclear leucocyte chemotactic response to leukotriene B4 in cystic fibrosis; Lawrence RH et al.; Evidence that leukotriene B4 (LTB4) is a significant inflammatory mediator in chronic pseudomonal respiratory disease was sought in adolescents and young adults with cystic fibrosis . Specific chemotaxis of peripheral blood polymorphonuclear leucocytes (PMN) was used as an indirect measure of remote in vivo exposure to LTB4 . PMN from 17 patients showed a significant decrease in chemotaxis to 10(-7)-10(-9) M LTB4, but normal responses to 10(-8) M n-formyl-methionyl-leucyl-phenylalanine and 4 mg/ml casein, when compared with 17 healthy age- and sex-matched controls . This result is consistent with chronic production of LTB4, and specific deactivation of circulating PMN receptors for LTB4 in patients with cystic fibrosis . Pharmacologic inhibition of LTB4 production in vivo may help elucidate its role in the pathogenesis of lung damage in cystic fibrosis.

Curr Microbiol, 1992 Aug, 25(2), 89 - 93
The effects of purified 25-kDa lipase from a clinical isolate of Pseudomonas cepacia in the lungs of rats; Lonon MK et al.; Nanogram quantities of a 25-kDa lipase purified from culture supernatants of Pseudomonas cepacia 90ee, a sputum isolate from a cystic fibrosis (CF) patient, were placed in the lungs of healthy rats . The resulting pathological changes included large amounts of proteinaceous exudate, the accumulation of polymorphonuclear leukocytes and red blood cells, and disorganization of alveolar structure . Pseudomonas cepacia 90ee immobilized in agar beads was also placed in the lungs of rats in a model of chronic infection . This resulted in bronchopneumonia and a milder inflammatory response than that elicited by the purified enzyme.

Biochim Biophys Acta, 1992 Jul 31, 1122(2), 189 - 95
A variety of catalases and bromoperoxidases in genus Pseudomonas and their characterization; Itoh N et al.; A survey of bromoperoxidase in some Pseudomonas strains revealed that they contain different types of bromoperoxidase, catalase-bromoperoxidase and catalase . Although all Pseudomonas strains exhibited catalase activity, the enzyme isolated from P . pyrrolnitrica was named catalase-bromoperoxidase, because it catalyzed not only a catalase reaction, but also the bromination of monochlorodimedone . Except heme-type catalase-bromoperoxidase, this strain contained four isoenzymes of general nonheme bromoperoxidase, and their molecular weights were about 73,000 . On the other hand, P . putida and P . aeruginosa had the usual heme-type catalase, but they differed in molecular weight and pI value . Both strains also had a nonheme bromoperoxidase which catalyzed the bromination of monochlorodimedone and aniline, and the molecular weight of each enzyme was 68,000 for P . putida and 86,000 for P . aeruginosa . Considering the results reported by Van Pee et al . {1} and Weisner et al . {2}, regarding the haloperoxidases of Pseudomonas, the genus was revealed to contain a wide variety of bromoperoxidase and catalase.

Biochemistry, 1992 Jul 28, 31(29), 6842 - 7
3-Hydroxy-3-methylglutaryl coenzyme A lyase: affinity labeling of the Pseudomonas mevalonii enzyme and assignment of cysteine-237 to the active site; Hruz PW et al.; Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) lyase is irreversibly inactivated by the reactive substrate analog 2-butynoyl-CoA . Enzyme inactivation, which follows pseudo-first-order kinetics, is saturable with a KI = 65 microM and a limiting k(inact) of 0.073 min-1 at 23 degrees C, pH 7.2 . Protection against inactivation is afforded by the competitive inhibitor 3-hydroxyglutaryl-CoA . Labeling of the bacterial enzyme with {1-14C}-2-butynoyl-CoA demonstrates that inactivation coincides with covalent incorporation of inhibitor, with an observed stoichiometry of modification of 0.65 per site . Avian HMG-CoA lyase is also irreversibly inactivated by 2-butynoyl-CoA with a stoichiometry of modification of 0.9 per site . Incubation of 2-butynoyl-CoA with mercaptans such as dithiothreitol results in the formation of a UV absorbance peak at 310 nm . Enzyme inactivation is also accompanied by the development of a UV absorbance peak at 310 nm indicating that 2-butynoyl-CoA modifies a cysteine residue in HMG-CoA lyase . Tryptic digestion and reverse-phase HPLC of the affinity-labeled protein reveal a single radiolabeled peptide . Isolation and sequence analysis of this peptide and a smaller chymotryptic peptide indicate that the radiolabeled residue is contained within the sequence GGXPY . Mapping of this peptide within the cDNA-deduced sequence of P . mevalonii HMG-CoA lyase {Anderson, D . H., & Rodwell, V . W . (1989) J . Bacteriol . 171, 6468-6472} confirms that a cysteine at position 237 is the site of modification . These data represent the first identification of an active-site residue in HMG-CoA lyase.

J Biol Chem, 1992 Jul 25, 267(21), 15064 - 70
Identification of the catalytically important histidine of 3-hydroxy-3-methylglutaryl-coenzyme A reductase; Darnay BG et al.; We identify His381 of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase as the basic residue functional in catalysis . The catalytic domain of 20 HMG-CoA reductases contains a single conserved histidine (His381 of the P . mevalonii enzyme) . Diethyl pyrocarbonate inactivated the P . mevalonii enzyme, and hydroxylamine partially restored activity . We changed His381 to alanine, lysine, asparagine, and glutamine . The mutant proteins were overexpressed, purified to homogeneity, and characterized . His381 mutant enzymes were not inactivated by diethyl pyrocarbonate . All four mutant enzymes exhibited wild-type crystal morphology and chromatographed on substrate affinity supports like wild-type enzyme . The mutant enzymes had low catalytic activity (Vmax 0.06-0.5% that of wild-type enzyme), but Km values approximated those for wild-type enzyme . For wild-type enzyme and mutant enzymes H381A, H381N, and H381Q, Km values at pH 8.1 were 0.45, 0.27, 3.7, and 0.71 mM {(R,S)-mevalonate}; 0.05, 0.03, 0.20, and 0.11 mM {coenzyme A}; 0.22, 0.14, 0.81, and 0.62 mM {NAD+} . Km values at pH 11 for wild-type enzyme and mutant enzyme H381K were 0.32 and 0.75 mM {(R,S)-mevalonate}; 0.24 and 0.50 mM {coenzyme A}; 0.15 and 1.23 mM {NAD+} . Both pK values for the enzyme-substrate complex increased relative to wild-type enzyme (by 1-2.5 pH units for pK1 and by 0.5-1.3 pH units for pK2) . For mutant enzyme H381K, the pK1 of 10.2 is consistent with lysine acting as a general base at high pH . His381 of P . mevalonii HMG-CoA reductase, and consequently the histidine of the consensus Leu-Val-Lys-Ser-His-Met-Xaa-Xaa-Asn-Arg-Ser motif of the catalytic domain of eukaryotic HMG-CoA reductases, thus is the general base functional in catalysis.

Biochem Pharmacol, 1992 Jul 22, 44(2), 397 - 400
Protection against tabun toxicity in mice by prophylaxis with an enzyme hydrolyzing organophosphate esters; Raveh L et al.; We demonstrate here the correlation between protection afforded by pretreatment alone with parathion hydrolase purified from Pseudomonas sp . against tabun toxicity in mice and the kinetic parameters which are assumed to determine the in vivo detoxification of tabun by the same enzyme . Results show that 15 and 22 micrograms of parathion hydrolase per animal conferred a protective ratio of 3.94 and 5.65 respectively, against tabun toxicity, without post-exposure treatment.

Kidney Int, 1992 Jul, 42(1), 61 - 8
Permeability of dialyzer membranes to TNF alpha-inducing substances derived from water bacteria; Lonnemann G et al.; Pro-inflammatory cytokine-inducing substances derived from cultured E . coli have previously been shown to pass across low-flux regenerated cellulosic dialyzer membranes . In the present study, a sterile filtrate of Pseudomonas maltophilia grown from standard bicarbonate dialysis fluid was used to test the permeability of various dialyzer membranes (regenerated cellulose, cellulose triacetate, polyacrylonitrile, polysulfone and polyamide) to TNF alpha-inducing bacterial substances . Pyrogen-free tissue culture medium (MEM) was recirculated for 60 minutes in the dialysate compartment of a closed-loop dialysis system, then P . maltophilia filtrate was added and recirculation was continued for a further hour . Samples from the dialysate (MEM) and the blood side (containing 10% human plasma in MEM) were incubated with donor mononuclear cells (MNC) for 18 hours and TNF alpha release was measured in MNC supernatants by radioimmunoassay . Five minutes after the addition of P . maltophilia filtrate, mean TNF alpha-inducing activity in the dialysate increased from (mean +/- SEM) 0.10 +/- 0.02 to 18.2 +2- 1.5 (ng/2.5 x 10(6) MNC/18 hr) . TNF alpha-inducing activity in the blood side increased with regenerated cellulose from 0.10 +/- 0.01 to 4.57 +/- 1.55 (N = 8; P less than 0.001); with cellulose triacetate from 0.20 +/- 0.05 to 0.44 +/- 0.10 (N = 5; P less than 0.05), and with polyacrylonitrile from 0.10 +/- 0.02 to 1.16 +/- 0.45 (N = 5; P less than 0.03) . No increased TNF alpha-inducing activity was observed in the blood side of polysulfone (N = 5) or polyamide dialyzers (N = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1992 Jul 1, 116(1), 1 - 6
Acetylornithine deacetylase, succinyldiaminopimelate desuccinylase and carboxypeptidase G2 are evolutionarily related; Boyen A et al.; The nucleotide (nt) sequence of the Escherichia coli argE gene, encoding the acetylornithine deacetylase (AO) subunit, has been established and corresponds to a 43-kDa (M(r) 42,320) polypeptide . The enzyme has been purified to near homogeneity and it appears to be a dimer consisting of two 43-kDa subunits . The amino acid sequence deduced from the nt sequence was compared to that of the subunit of E . coli succinyldiaminopimelate desuccinylase (the dapE gene product involved in the diaminopimelate pathway for lysine biosynthesis), since both enzymes share functional and biochemical features . Significant similarity covering the entire sequence allows us to infer a common origin for both deacylases . This homology extends to the Pseudomonas sp . G2 carboxypeptidase (G2CP); this or a functionally related enzyme may be responsible for the minor AO activity found in organisms relying on ornithine acetyltransferase for ornithine biosynthesis.

Clin Infect Dis, 1992 Jul, 15(1), 163 - 9
Neurological melioidosis: seven cases from the Northern Territory of Australia; Woods ML 2nd et al.; Pseudomonas pseudomallei, which causes melioidosis, is most commonly associated with pulmonary infection . We describe seven patients who developed a neurological syndrome as the predominant manifestation of melioidosis: this syndrome was characterized by peripheral motor weakness (mimicking Guillain-Barre syndrome), brain-stem encephalitis, aseptic meningitis, and respiratory failure . Neurological melioidosis occurred in the absence of demonstrable foci of infection in the central nervous system (CNS) in five of six patients whose cerebrospinal fluid was available for culture . Computed tomography and magnetic resonance imaging of the brain and spinal cord of these patients were not suggestive of pyogenic infection, although the latter procedure detected brain-stem encephalitis . Autopsy findings in one case confirmed brain-stem encephalitis without evidence of direct bacterial infection . The clinical presentation of neurological melioidosis includes features of an exotoxin-induced neurological syndrome, with profound neurological disease occurring in the absence of apparent direct infection of the CNS . This syndrome appears to be a newly recognized clinical presentation of melioidosis.

Mikrobiyol Bul, 1992 Jul, 26(3), 281 - 3
{Capacity of fish media for the production of Pseudomonas pigments}; Kantarci G; Fish mediums B1 and B2, which modified King A base (B1) and modified King B base (B2) were examined for pigment production of P.aeruginosa strains and compared with King A and King B Mediums . Pyocyanin production on B1 was found better than on King A medium . Pyoverdin (Fluorescein) production on B2 was observed as a lighter yellow green than King B Medium . Fluorescence on B2 was recognized easier than King B Medium . As a result of this study, Fish Medium B1 and B2 can be used easily for examination of pigments of Pseudomonas strains . The other advantage of fish medium is, that, it can be produced easily in our country.

Mol Plant Microbe Interact, 1992 Jul-Aug, 5(4), 322 - 9
Identification of a lysA-like gene required for tabtoxin biosynthesis and pathogenicity in Pseudomonas syringae pv . tabaci strain PTBR2.024; Engst K et al.; Pseudomonas syringae pv . tabaci strain PTBR2.024 produces tabtoxin and causes wildfire disease on tobacco and green bean . PTBR7.000, a Tn5 mutant of PTBR2.024, does not produce tabtoxin, is nonpathogenic on tobacco, and is prototrophic . A 3-kb fragment from a genomic library of the parent strain PTBR2.024 complemented both mutant phenotypes . This 3-kb fragment contains two open reading frames (ORFs), ORF1 and ORF2, and two truncated ORFs, ORF3 and ORF4 . The Tn5 insert in PTBR7.000 was mapped to ORF2, and complementation studies showed that an intact ORF2 was sufficient to restore tabtoxin production and pathogenicity . The deduced amino acid sequences of ORF2 and truncated ORF3 contain significant homology to bacterial lysine biosynthetic enzymes, diaminopimelate decarboxylase, and delta 1-piperidine-2,6-dicarboxylate succinyl transferase, respectively . ORF2, however, is not required for lysine biosynthesis . We designated the sequence corresponding to ORF2 as gene tabA and propose that the product of tabA is an enzyme in the tabtoxin biosynthetic pathway that recognizes a substrate analogue of a compound in the lysine biosynthetic pathway.

Bioconjug Chem, 1992 Jul-Aug, 3(4), 302 - 7
Cytotoxicity of chimeric (human-murine) monoclonal antibody BR96 IgG, F(ab')2, and Fab' conjugated to Pseudomonas exotoxin; Siegall CB et al.; We have made antigen-specific cytotoxic reagents by conjugating the chimeric antibody BR96 (chiBR96) to Pseudomonas exotoxin A (PE), as either native PE or a truncated form (LysPE40) devoid of the cell-recognition region (domain I) . PE kills cells by ADP-ribosylation of elongation factor 2, thereby inhibiting protein synthesis . Chimeric BR96 immunotoxins were constructed by chemical conjugation of the toxin to Fab', F(ab')2, and intact IgG and purified by anion-exchange and gel-filtration chromatography . Chimeric BR96 {IgG and F(ab')2} immunotoxins were cytotoxic against tumor cell lines displaying the BR96 antigen, with EC50 values ranging from 0.1 to 110 pM . Immunotoxins constructed with chiBR96 Fab' were 50-100-fold less cytotoxic . Competition analysis showed that the immunotoxins were specifically active through their BR96 antigen-binding ability . The binding of chiBR96-PE and chiBR96-LysPE40 to antigen was equivalent to that of BR96 itself and these immunotoxins were found to internalize very rapidly, displaying 90% of their cytotoxicity within 1 h . Binding assays determined that chiBR96 F(ab')2-LysPE40 bound as well as chiBR96-LysPE40; however, chiBR96 Fab'-LysPE40 bound 20-fold less efficiently . The chiBR96 Fab'-LysPE40 internalized similarly to the F(ab')2 or the IgG immunotoxins . Therefore, the chiBR96 Fab'-LysPE40 immunotoxin is less cytotoxic toward target cells because of reduced antigen binding . This is may be due to the monovalent nature of chiBR96 Fab'-LysPE40 . This study shows that the monoclonal antibody chiBR96-Pseudomonas exotoxin A immunotoxins can be effective at inhibiting protein synthesis in target cells.

J Hosp Infect, 1992 Jul, 21(3), 199 - 204
Pseudomonas cepacia pulmonary infection in adults with cystic fibrosis: is nosocomial acquisition occurring?
Taylor RF, Dalla Costa L, Kaufmann ME, Pitt TL, Hodson ME.
Ribotyping of 25 isolates of Pseudomonas cepacia taken from the sputum of 21 adults with cystic fibrosis (CF) who were registered at the Royal Brompton Hospital between 1987 and 1991, revealed that seven patients (33.3%) shared strains of a similar ribotype pattern with others, while 14 patients (63.7%) harboured strains unique to each individual . Constancy of sputum strain carriage was seen in two of three patients sampled twice over a 3-month period . Although no evidence for patient-to-patient transmission of P . cepacia within this group of patients was found, the fact that one third of CF patients shared strains of the same ribotype with others, suggests that nosocomial acquisition of this organism may have occurred.

Curr Microbiol, 1992 Jul, 25(1), 25 - 9
High-frequency electroporation and maintenance of pUC- and pBR-based cloning vectors in Pseudomonas stutzeri; Pemberton JM et al.; A number of Escherichia coli cloning vectors, based on ColE1-like replicons, were shown to be maintained in Pseudomonas stutzeri ATCC 17588 . A restrictionless mutant of P . stutzeri was isolated, and this strain was used to develop an efficient electroporation system . With the E . coli cloning vector pHSG298, transformation frequencies of up to 2 x 10(7) transformants/micrograms DNA were achieved . This frequency is comparable to that obtained for CaCl2-mediated transformation of E . coli; thus, direct cloning of DNA into P . stutzeri is feasible . As will be discussed, this may prove useful for cloning DNA from high mol% G + C genera in cases in which E . coli is not a suitable heterologous cloning host.

Proc Natl Acad Sci U S A, 1992 Jul 1, 89(13), 5867 - 71
Recombinant anti-erbB2 immunotoxins containing Pseudomonas exotoxin; Batra JK et al.; Immunotoxins were made using five different murine monoclonal antibodies to the human erbB2 gene product and LysPE40, a 40-kDa recombinant form of Pseudomonas exotoxin (PE) lacking its cell-binding domain . All five conjugates were specifically cytotoxic to cancer cell lines overexpressing erbB2 protein . The most active conjugate was e23-LysPE40, generated by chemical crosslinking of anti-erbB2 monoclonal antibody e23 to LysPE40 . In addition, a recombinant immunotoxin, e23(Fv)PE40, was constructed that consists of the light-chain variable domain of e23 connected through a peptide linker to its heavy-chain variable domain, which in turn is fused to PE40 . The recombinant protein was made in Escherichia coli, purified to near homogeneity, and shown to selectively kill cells expressing the erbB2 protooncogene . To improve the cytotoxic activity of e23(Fv)PE40, PE40 was replaced with a variant, PE38KDEL, in which the carboxyl end of PE is changed from Arg-Glu-Asp-Leu-Lys to Lys-Asp-Glu-Leu and amino acids 365-380 of PE are deleted . The e23(Fv)PE38KDEL protein inhibits the growth of tumors formed by the human gastric cancer cell line N87 in immunodeficient mice.

Mol Plant Microbe Interact, 1992 Jul-Aug, 5(4), 330 - 9
Cloning and heterologous expression of the phenazine biosynthetic locus from Pseudomonas aureofaciens 30-84; Pierson LS 3rd et al.; Pseudomonas aureofaciens strain 30-84 suppresses take-all disease of wheat caused by Gaeumannomyces graminis var . tritici . Three antibiotics, phenazine-1-carboxylic acid, 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxyphenazine, were responsible for disease suppression . Tn5-induced mutants deficient in production of one or more of the antibiotics (Phz-) were significantly less suppressive than the parental strain . Cosmids pLSP259 and pLSP282 from a genomic library of strain 30-84 restored phenazine production and fungal inhibition to 10 different Phz- mutants . Sequences required for production of the phenazines were localized to a segment of approximately 2.8 kilobases that was present in both cosmids . Expression of this locus in Escherichia coli required the introduction of a functional promoter, was orientation-specific, and resulted in the production of all three phenazine antibiotics . These results strongly suggest that the cloned sequences encode a major portion of the phenazine biosynthetic pathway.

J Gen Microbiol, 1992 Jul, 138 ( Pt 7), 1337 - 43
Stability, frequency and multiplicity of transposon insertions in the pyoverdine region in the chromosomes of different fluorescent pseudomonads; Cornelis P et al.; Tn5 mutagenesis of different fluorescent pseudomonads was achieved by conjugational transfer of the suicide vector pSUP 10141 . Pyoverdine negative (Pvd-) mutants were detected by the absence of fluorescence on King's B medium and by their inability to grow in the presence of the iron chelator EDDHA {ethylenediamine di(o-hydroxyphenylacetic acid)} . In P . fluorescens ATCC 17400 and three rhizosphere isolates (one P . putida and two P . fluorescens), the percentage of Pvd- mutants ranged between 0 and 0.54% . In a P . chlororaphis rhizosphere isolate, this percentage was higher (4%) . In these mutants both of the Tn5 antibiotic resistances (Km and Tc) were stable and the transposon could be detected by hybridization . In Pvd- mutants of P . fluorescens ATCC 17400, the transposon was found to be inserted twice in the chromosome while single insertions were detected in the DNA of other, randomly tested mutants . In P . aeruginosa PAO1, where 13.1% of the mutants were Pvd-, both antibiotic resistances were rapidly lost and accordingly no transposon insertion could be detected by hybridization . However, the Pvd- phenotype was generally stable in these mutants . The plasmid pNK862 containing a mini-Tn10 transposon was introduced by electroporation into P . aeruginosa PAO1 and Kmr mutants were recovered, 89% of which were Pvd- and confirmed to be P . aeruginosa by PCR amplification of the P . aeruginosa lipoprotein gene . The mini-Tn10 insertions were also found to be unstable in PAO1.

J Biol Chem, 1992 Jun 25, 267(18), 12420 - 3
The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes Pseudomonas exotoxin A; Kounnas MZ et al.; The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2 MR/LRP) is a large cell-surface glycoprotein consisting of a 515-kDa and an 85-kDa polypeptide; this receptor is thought to be responsible for the binding and endocytosis of activated alpha 2-macroglobulin and apoE-enriched beta-very low density lipoprotein . A similar high molecular weight glycoprotein has been identified as a potential receptor for Pseudomonas exotoxin A (PE) . We demonstrate that the alpha 2 MR/LRP and the PE-binding glycoprotein have a similar mobility upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and are immunologically indistinguishable . Furthermore, affinity-purified alpha 2 MR/LRP binds specifically to PE but not to a mutant toxin defective in its ability to bind cells . The 39-kDa receptor-associated protein, which blocks binding of ligands to alpha 2 MR/LRP, also prevents binding and subsequent toxicity of PE for mouse fibroblasts . The concentration of receptor-associated protein that was required to reduce binding and toxicity to 50% was approximately 14 nM, a value virtually identical to the KD measured for the interaction of receptor-associated protein with the purified receptor . Overall, the studies strongly suggest that the alpha 2 MR/LRP is responsible for internalizing PE.

Biochemistry, 1992 Jun 23, 31(24), 5605 - 10
Isolation and characterization of the three polypeptide components of 4-chlorobenzoate dehalogenase from Pseudomonas sp . strain CBS-3; Chang KH et al.; The three genes encoding the 4-chlorobenzene dehalogenase polypeptides were excised from a Pseudomonas sp . CBS-3 DNA fragment and separately cloned and expressed in Escherichia coli . The three enzymes were purified from the respective subclones by using an ammonium sulfate precipitation step followed by one or two column chromatographic steps . The 4-chlorobenzoate:coenzyme A ligase was found to be a homodimer (57-kDa subunit size), to require Mg2+ (Co2+ and Mn2+ are also activators) for activity, and to turn over MgATP (Km = 100 microM), coenzyme A (Km = 80 microM), and 4-chlorobenzoate (Km = 9 microM) at a rate of 30 s-1 at pH 7.5 and 25 degrees C . Benzoate, 4-bromobenzoate, 4-iodobenzoate, and 4-methylbenzoate were shown to be alternate substrates while 4-hydroxybenzoate, 4-aminobenzoate, 2-aminobenzoate, 2,3-dihydroxybenzoate, 4-coumarate, palmate, laurate, caproate, butyrate, and phenylacetate were not substrate active . The 4-chlorobenzoate-coenzyme A dehalogenase was found to be a homotetramer (30 kDa subunit size) to have a Km = 15 microM and kcat = 0.3 s-1 at pH 7.5 and 25 degrees C and to be catalytically inactive toward hydration of crotonyl-CoA, alpha-methylcrotonyl-CoA, and beta-methylcrotonyl-CoA . The 4-hydroxybenzoate-coenzyme A thioesterase was shown to be a homotetramer (16 kDa subunit size), to have a Km = 5 microM and kcat = 7 s-1 at pH 7.5 and 25 degrees C, and to also catalyze the hydrolyses of benzoyl-coenzyme A and 4-chlorobenzoate-coenzyme A . Acetyl-coenzyme A, hexanoyl-coenzyme A, and palmitoyl-coenzyme A were not hydrolyzed by the thioesterase.

Biochemistry, 1992 Jun 23, 31(24), 5594 - 604
Ancestry of the 4-chlorobenzoate dehalogenase: analysis of amino acid sequence identities among families of acyl:adenyl ligases, enoyl-CoA hydratases/isomerases, and acyl-CoA thioesterases; Babbitt PC et al.; We have deduced the nucleotide sequence of the genes encoding the three components of 4-chlorobenzoate (4-CBA) dehalogenase from Pseudomonas sp . CBS-3 and examined the origin of these proteins by homology analysis . Open reading frame 1 (ORF1) encodes a 30-kDa 4-CBA-coenzyme A dehalogenase related to enoyl-coenzyme A hydratases functioning in fatty acid beta-oxidation . ORF2 encodes a 57-kDa protein which activates 4-CBA by acyl adenylation/thioesterification . This 4-CBA:coenzyme A ligase shares significant sequence similarity with a large group of proteins, many of which catalyze similar chemistry in beta-oxidation pathways or in siderophore and antibiotic synthetic pathways . These proteins have in common a short stretch of sequence, (T,S)(S,G)G(T,S)(T,E)G(L,X)PK(G,-), which is particularly highly conserved and which may represent an important new class of "signature" sequence . We were unable to find any proteins homologous in sequence to the 16-kDa 4-hydroxybenzoate-coenzyme A thioesterase encoded by ORF3 . Analysis of the chemistry and function of the proteins found to be structurally related to the 4-CBA:coenzyme A ligase and the 4-CBA-coenzyme A dehalogenase supports the proposal that they evolved from a beta-oxidation pathway.

Vet Rec, 1992 Jun 13, 130(24), 525 - 9
An outbreak of melioidosis in imported primates in Britain; Dance DA et al.; An outbreak of melioidosis, a bacterial infection caused by Pseudomonas pseudomallei, was identified in a batch of feral cynomolgus monkeys (Macaca fascicularis) imported to Britain from the Philippines . Thirteen confirmed or possible cases occurred among a batch of 50 animals . Subsequent investigations revealed that the infection was uncommon among imported primates from a variety of sources, although three other cases were identified in monkeys imported from Indonesia . The majority of the affected monkeys had splenic abscesses, and hepatic abscesses and infections of the soft tissues and skin were also frequently observed . Most of the infected animals had no clinical signs despite extensive abscesses, and the presence of infection was only suspected when they were shown to have serum antibodies to P pseudomallei by an enzyme-linked immunosorbent assay . Although there was no evidence of cross infection of other animals or human handlers, this outbreak is a reminder of the dangers of working with wild-caught primates and the potential for the establishment of environmental foci of melioidosis.

J Clin Microbiol, 1992 Jun, 30(6), 1583 - 4
Pseudomonas mendocina, an environmental bacterium isolated from a patient with human infective endocarditis; Aragone MR et al.; Pseudomonas mendocina has been isolated from soil and water samples . Although it has been recovered from some human clinical samples, its pathogenic role has not yet been documented . We report the first known case of endocarditis in humans due to P . mendocina.

Appl Environ Microbiol, 1992 Jun, 58(6), 1986 - 91
Biological suppression of potato ring rot by fluorescent pseudomonads; de la Cruz AR et al.; Three strains of fluorescent pseudomonads (IS-1, IS-2, and IS-3) isolated from potato underground stems with roots showed in vitro antibiosis against 30 strains of the ring rot bacterium Clavibacter michiganensis subsp . sepedonicus . On the basis of morphological and biochemical tests and fatty acid analysis, IS-1 and IS-2 were identified as Pseudomonas aureofaciens and IS-3 was identified as P . fluorescens biovar III . IS-1 was the most inhibitory to C . michiganensis subsp . sepedonicus strains in vitro, followed by IS-3 and IS-2 . Suppression of ring rot by these antagonists was demonstrated in greenhouse trials with stem-cultured potato (cv . Russet Burbank) seedlings . Although each antagonist significantly reduced C . michiganensis subsp . sepedonicus populations, only IS-1 reduced infection by C . michiganensis subsp . sepedonicus . In a second experiment, treatment with IS-1 (10(9) CFU/ml) significantly reduced ring rot infection by 23.4 to 26.7% after 5 to 8 weeks . The average C . michiganensis subsp . sepedonicus population was also significantly reduced by 50 to 52% . Application of different combinations of antagonist strains was not more effective than single-strain treatment.

Eur J Biochem, 1992 Jun 1, 206(2), 441 - 52
Crystal structure of NAD-dependent formate dehydrogenase; Lamzin VS et al.; The ternary complex of NAD-dependent formate dehydrogenase (FDH) from the methylotrophic bacterium Pseudomonas sp . 101 (enzyme-NAD-azide) has been crystallised in the space group P2(1)2(1)2(1) with cell dimensions a = 11.60 nm, b = 11.33 nm, c = 6.34 nm . There is 1 dimeric molecule/asymmetric unit . An electron density map was calculated using phases from multiple isomorphous replacement at 0.30 nm resolution . Four heavy atom derivatives were used . The map was improved by solvent flattening and molecular averaging . The atomic model, including 2 x 393 amino acid residues, was refined by the CORELS and PROLSQ packages using data between 1.0 nm and 0.30 nm excluding structure factors less than 1 sigma . The current R factor is 27.1% and the root mean square deviation from ideal bond lengths is 4.2 pm . The FDH subunit is folded into a globular two-domain (coenzyme and catalytic) structure and the active centre and NAD binding site are situated at the domain interface . The beta sheet in the FDH coenzyme binding domain contains an additional beta strand compared to other dehydrogenases . The difference in quaternary structure between FDH and the other dehydrogenases means that FDH constitutes a new subfamily of NAD-dependent dehydrogenases: namely the P-oriented dimer . The FDH nucleotide binding region of the structure is aligned with the three dimensional structures of four other dehydrogenases and the conserved residues are discussed . The amino acid residues which contribute to the active centre and which make contact with NAD have been identified.

J Bacteriol, 1992 Jun, 174(11), 3499 - 507
Plant and environmental sensory signals control the expression of hrp genes in Pseudomonas syringae pv . phaseolicola; Rahme LG et al.; The hrp genes of Pseudomonas syringae pv . phaseolicola control the development of primary disease symptoms in bean plants and the elicitation of the hypersensitive response in resistant plants . We examined the expression of the seven operons located in the 22-kb hrp cluster (L . G . Rahme, M . N . Mindrinos, and N . J . Panopoulos, J . Bacteriol . 173:575-586, 1991) in planta and in vitro under different physiological and nutritional conditions by using chromosomally located hrp::inaZ reporter fusions . We show that (i) a plant signal(s) is specifically required for the induction of the seven hrp operons, during both compatible and incompatible interactions; (ii) hrpL and hrpRS are regulated by different mechanisms in planta and in vitro; and (iii) expression of individual hrp loci is differentially affected by pH, osmotic strength, and type of carbon source: hrpAB, hrpC, and hrpD were downregulated similarly by osmolarity, pH, and certain carbon sources; hrpE expression was affected strongly by pH and carbon substrate and slightly by osmolarity; and hrpF was not substantially affected by any of these factors . These findings suggest complex signaling mechanisms taking place during plant-pathogen interactions.

Cancer Res, 1992 Jun 1, 52(11), 3189 - 93
Antitumor effects of B3-PE and B3-LysPE40 in a nude mouse model of human breast cancer and the evaluation of B3-PE toxicity in monkeys; Pai LH et al.; B3 is a tumor-reactive monoclonal antibody (mAb) that binds to a limited number of normal tissues . Immunotoxins made with B3 coupled to either Pseudomonas exotoxin (PE) or recombinant forms of PE with a deletion of the cell-binding domain (LysPE40) have been shown to cause complete tumor regression in nude mice bearing a rapidly growing A431 (L . H . Pai et al., Proc . Natl . Acad . Sci . USA, 88: 3358-3362, 1991) human epidermoid carcinoma . In this study we show that an immunotoxin composed of mAb B3 when chemically coupled to LysPE40 (B3-LysPE40) led to complete regression of a slowly growing breast cancer, MCF-7, in nude mice when given i.v . every other day for five doses . mAb B3 coupled to native PE also produced significant regression of the MCF-7 tumor . The reactivity of mAb B3 was evaluated using an immunohistochemical method on the two responsive tumors, MCF-7 and A431, and compared with a typical human colon carcinoma specimen that has B3 antigen on its surface . The results showed that both A431 and MCF-7 xenograft tumors have similar reactivity to B3 when compared with the human colon carcinoma specimen . To evaluate the toxicity of B3-PE in primates, Cynomolgus monkeys received escalating doses of B3-PE i.v . on Days 1, 3, and 5 . Based on antibody localization studies using frozen sections of normal human and monkey tissue, gastric, trachea, and bladder mucosal injury could have occurred . However, no clinical signs of injury or histological damage to these organs were seen at the doses administered . Chemical hepatitis due to PE was transient and well tolerated at doses up to 50 micrograms/kg for three doses . The lethal dose was about 100 micrograms/kg, and the cause of death was liver necrosis, as shown by necropsy . We conclude that mAb B3, when coupled to PE40 or PE, can produce strong antitumor activity in vivo . The similar level of reactivity of the B3 antibody in our tumor models with a surgical specimen of a human colon carcinoma and the toxicity study in monkeys indicate that therapeutic doses of B3-PE and B3-LysPE40 can be delivered without causing toxicity to normal organs that express B3 antigen . Although both B3-PE and B3-LysPE40 have antitumor activity in nude mice bearing a human xenograft, B3-LysPE40 is better tolerated and should be further evaluated as a therapeutic agent for cancer patients.

Hua Xi Yi Ke Da Xue Xue Bao, 1992 Jun, 23(2), 156 - 9
{Isolation and identification of a strain of Pseudomonas SP . producing insoluble glucan hydrolase}; He G et al.; A strain, CIB871, isolated from the natural world can hydrolyze insoluble glucan(IG) produced by S . mutans . This organism has been identified to be a strain of Pseudomonas SP . It grows well at 30 degrees C . The optimum medium for producing insoluble glucan hydrolase (IGase) is composed of 0.3% peptone, 0.03% IG and M1 salt solution (NH4)2 SO4 1mg/ml, MgSO4 50 micrograms/ml, FeCl2 50 micrograms/ml, K2HPO4 0.5mg/ml) . The IGase production reached maximum when cultured for 65-70 hours.

Biochimie, 1992 Jun, 74(6), 561 - 4
Kinetics and stability of delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni in the system of reverse micelles of aerosol OT in isooctane; Levashov AV et al.; Partially purified delta 5-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni was studied kinetically after solubilization in reverse micelles of aerosol OT (AOT) in isooctane and water, as regards its application to biotechnology . With delta 5,10-estren-17 beta-ol-3-one as a substrate, KSI displays an enzyme activity in the micellar system but a low stability . In the presence of urea, the enzyme is, however, stable . Kinetic parameters of the stabilized enzyme are highly sensitive to both the hydration degree of the surfactant and its concentration . The hypothesis of the geometric correspondence of a non-spherical enzyme and spherical micellar matrix is considered.

Infect Immun, 1992 Jun, 60(6), 2402 - 8
Prolonged elevation of interleukin-8 and interleukin-6 concentrations in plasma and of leukocyte interleukin-8 mRNA levels during septicemic and localized Pseudomonas pseudomallei infection; Friedland JS et al.; Patients suffering from serious bacterial infection present to the hospital after early inflammatory events, such as release of tumor necrosis factor (TNF), have been initiated . The role of other cytokines, such as interleukin-8 (IL-8), a neutrophil chemoattractant and activator, in the pathophysiology of human sepsis is not well characterized, and there are only limited data on IL-6 . We studied serial concentrations of TNF, IL-6 (involved in the acute-phase response), and IL-8 in plasma and leukocyte levels of mRNA for these cytokines in patients with localized and septicemic Pseudomonas pseudomallei infection on admission to the hospital and during a prolonged recovery phase (up to 30 days) . Of 18 patients, 8 had detectable plasma IL-8 and all had raised plasma IL-6 concentrations . In patients who died median initial concentration of IL-8 (167 pg/ml; range, 97 to 362 pg/ml) and IL-6 (4,800 pg/ml; range, 60 to 9,245 pg/ml) in plasma were higher than those in survivors (P less than 0.008 and P = 0.007, respectively) . Septic patients who survived and patients with localized disease had similar cytokine levels . Plasma IL-8 and IL-6 concentrations were elevated throughout the inpatient period of recovery . Circulating leukocytes contained mRNA for IL-8 but not for IL-6 and TNF, and they may secrete IL-8 . An elevated plasma IL-6 concentration (greater than 1,000 pg/ml) had 75% mortality) was the best predictor of mortality in P . pseudomallei sepsis . Fifty percent of patients with detectable plasma IL-8 concentrations died . In contrast, plasma TNF bioactivity did not relate to outcome; 75% of patients who did never had detectable plasma TNF activity.

J Bacteriol, 1992 Jun, 174(11), 3461 - 6
Identification and sequencing of a gene encoding a hydantoin racemase from the native plasmid of Pseudomonas sp . strain NS671; Watabe K et al.; DNA fragments containing the genes involved in the conversion of 5-substituted hydantoins to their corresponding L-amino acids have been cloned from the 172-kb native plasmid (pHN671) of Pseudomonas sp . strain NS671 . The largest recombinant plasmid, designated pHPB14, encoded the ability to convert D-5-substituted hydantoins to the corresponding L-amino acids, whereas the smallest one, designated pHPB12, encoded the ability to convert them to their corresponding N-carbamyl-D-amino acids . Restriction analysis suggested that the inserts of both recombinant plasmids are derived from the identical portion in pHN671 and that the insert of pHPB14, compared with that of pHPB12, has an extra 5.3 kb in length . DNA sequencing revealed that pHPB14 contains two additional complete open reading frames, designated ORF5 and hyuE . Analysis of deletion derivatives of pHPB14 indicated that hyuE is required for the ability to produce L-amino acids from the corresponding D-5-substituted hydantoins, but ORF5 is not . Cells of Escherichia coli transformed with a plasmid containing hyuE were capable of racemizing different 5-substituted hydantoins, indicating that hyuE is a gene encoding a hydantoin racemase.

FEMS Microbiol Lett, 1992 May 15, 72(1), 49 - 55
Purification and characterisation of an NAD(+)-dependent secondary alcohol dehydrogenase from Pseudomonas maltophilia MB11L; Britt AJ et al.; A constitutive NAD(+)-linked alcohol dehydrogenase was purified 338-fold from cells of Pseudomonas maltophilia MB11L grown on glucose . Maximum activity was observed with cyclic and linear secondary alcohols, with little activity seen against primary or aromatic alcohols . Substrate oxidation activity was maximal at pH 10.0, while substrate reduction was optimal at pH 4.5 . The Km values for propan-2-ol, NAD+ and acetone were 87, 413 and 143 microM respectively . The enzyme is a tetramer with subunit Mr of approximately 44,000 . It has an isoelectric point of 4.75, and was inhibited by chelating agents, thiol reagents and certain metal ions.

Biochem Biophys Res Commun, 1992 May 15, 184(3), 1386 - 92
Specific activation of a tyrosine----glycine mutant of delta 5-3-ketosteroid isomerase by phenols; Brooks B et al.; A key unknown still to be explored concerning the mechanism of delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni is the extent of the proton transfer between tyrosine-14 of the enzyme and the C-3 carbonyl oxygen of the steroid substrate . This report is a preliminary study of a system we are developing to allow us eventually to use a Bronsted analysis to measure this transfer . We describe the construction of an expression vector and tyrosine-14----glycine-14 mutant of the enzyme and its specific activation, in the manner of chemical rescue, by a variety of phenolic compounds . We suggest that the binding region of phenol is very tight and that the level of activation may be a result of steric constraints as well as of differences in the pKa' of the phenol.

J Biol Chem, 1992 May 5, 267(13), 9194 - 201
Topology of the membrane-bound alkane hydroxylase of Pseudomonas oleovorans; van Beilen JB et al.; The Pseudomonas oleovorans alkane hydroxylase is an integral cytoplasmic membrane protein that is expressed and active in both Escherichia coli and P . oleovorans . Its primary sequence contains eight hydrophobic stretches that could span the membrane as alpha-helices . The topology of alkane hydroxylase was studied in E . coli using protein fusions linking different amino-terminal fragments of the alkane hydroxylase (AlkB) to alkaline phosphatase (PhoA) and to beta-galactosidase (LacZ) . Four AlkB-PhoA fusions were constructed using transposon TnphoA . Site-directed mutagenesis was used to create PstI sites at 12 positions in AlkB . These sites were used to create AlkB-PhoA and AlkB-LacZ fusions . With respect to alkaline phosphatase and beta-galactosidase activity each set of AlkB-PhoA and AlkB-LacZ fusions revealed the expected complementary activities . At three positions, PhoA fusions were highly active, whereas the corresponding LacZ fusions were the least active . At all other positions the PhoA fusions were almost completely inactive, but the corresponding LacZ fusions were highly active . These data predict a model for alkane hydroxylase containing six transmembrane segments . In this model the amino terminus, two hydrophilic loops, and a large carboxyl-terminal domain are located in the cytoplasm . Only three very short loops near amino acid positions 52, 112, and 251 are exposed to the periplasm.

Appl Environ Microbiol, 1992 May, 58(5), 1780 - 3
Evidence for a correlation between auxin production and host plant species among strains of Pseudomonas syringae subsp . savastanoi; Gardan L et al.; Auxin production by 131 strains of Pseudomonas syringae subsp . savastanoi was investigated with the aim of looking for correlations among this characteristic and the origin of the strains, the types of symptoms, and the host plant . Most of the P.syringae subsp . savastanoi strains, except those isolated from ash, produced auxin and harbored iaa genes . Among ash strains, which were pathogenic only on ash, only 2 out of 33 were found to produce auxin and to harbor iaa genes.

Appl Environ Microbiol, 1992 May, 58(5), 1776 - 9
Degradation of triglycerides by a pseudomonad isolated from milk: molecular analysis of a lipase-encoding gene and its expression in Escherichia coli; Johnson LA et al.; Psychrotrophic lipolytic bacteria represent a significant problem in the storage of refrigerated dairy products . A lipase-encoding gene has been cloned and characterized from a highly lipolytic strain of Pseudomonas . The nucleotide sequence of the gene predicts a polypeptide of M(r) 49,905, which was identified when the gene was expressed in Escherichia coli.

Mol Gen Genet, 1992 May, 233(1-2), 42 - 8
Processing by OmpT of fusion proteins carrying the HlyA transport signal during secretion by the Escherichia coli hemolysin transport system; Hanke C et al.; A fusion gene (ces-hlyAs) was constructed by ligating the genetic information for the C-terminal 60 amino acids (hlyAs) of Escherichia coli hemolysin (HlyA) to the ces gene for a cholesterol esterase/lipase (CE) from a Pseudomonas species . Part (about 30%) of the expressed fusion protein CE-HlyAs was secreted in E . coli carrying hlyB and hlyD genes . Following the insertion between the reporter gene and hlyAs of a linker sequence that contains the information for potential cleavage sites for the outer membrane protease OmpT, two different fusion proteins (PhoA-HlyAs and CE-HlyAs) were shown to be cleaved by OmpT between the two parts during HlyB/HlyD-mediated secretion . Processed PhoA and CE accumulated in the supernatant . The efficiency of cleavage by OmpT was considerably improved by increased ompT gene dose . It was further shown that OmpT preferentially recognizes potential cleavage sites within the linker sequence.

J Histochem Cytochem, 1992 May, 40(5), 711 - 21
Validation of the biotinyl ligand-avidin-gold technique; Morris RE et al.; We demonstrate here that the intracellular routing of biotinylated ligands was not affected by the attachment of streptavidin gold colloids so long as the electron-dense marker was added after the biotinyl ligand-receptor interaction had occurred . The binding, internalization, and intracellular routing of three different biotinyl ligands were followed in mouse LM fibroblasts . The biotinyl (B) ligands included B-choleragenoid (B-CTd), B-wheat germ agglutinin (B-WGA), and B-Pseudomonas exotoxin A (B-PE) . All three ligands showed distinct intracellular trafficking patterns . B-WGA and B-PE entered via clathrin-coated pits, whereas B-CTd did not . After entry, B-CTd was routed to the lysosomal compartment without involvement of the Golgi . Although B-PE and B-WGA were also routed to the lysosomal compartment, a significant portion of these two ligands was observed in association with the Golgi . B-WGA, however, remained in the endosomal and Golgi compartments longer than did B-PE . We also monitored the internalization and routing of native PE by an indirect immunoperoxidase technique done in conjunction with saponin solubilization . The results corroborated the observations with the biotinyl-PE-streptavidin-gold method . In contrast, biotinyl-PE added to streptavidin-gold before addition to LM cells was poorly internalized and routed aberrantly . From these observations we conclude that the biotinyl ligand-avidin-gold technique is a valid method for following the binding, internalization, and intracellular routing of ligands.

Anal Biochem, 1992 May 1, 202(2), 331 - 6
The use of a layering technique for enhancing stability of lyophilized reagents; Ramsay JR et al.; An enzyme-mediated assay has been developed for the measurement of salicylate using salicylate monooxygenase purified from Pseudomonas cepacia ATCC 29351 . Two assay formulations were produced, based on either a multiple-reagent or a single-reagent formulation, to allow sufficient flexibility for automated use . The multiple-reagent formulation was especially suited to diagnostic laboratories performing infrequent manual salicylate estimation where stability of the reconstituted reagent is of paramount importance . This was achieved by preparing the enzyme and color reagents in separate vials, so keeping the enzyme at a stable pH . For more frequent assay use where a reconstituted reagent shelf life was less important, the single-reagent system offers advantages of convenience . However, the working reagent required a pH of 10.0 upon reconstitution . Although the enzyme was sufficiently active at this pH to give a reliable assay, its storage stability was poor at pH 10.0, preventing lyophilization of the reagent at a pH suitable for immediate use on reconstitution . This incompatibility was overcome by use of a layering technique . The enzyme was separated from the buffering solution in the same vial by freezing the buffering solution and then overlayering with the enzyme reagent prior to a second freezing cycle and subsequent freeze drying.

Appl Environ Microbiol, 1992 May, 58(5), 1760 - 3
Pseudomonas cepacia suppression of sunflower wilt fungus and role of antifungal compounds in controlling the disease; McLoughlin TJ et al.; In a field experiment, Pseudomonas cepacia J82rif and J51rif increased sunflower emergence in the presence of the fungus Sclerotinia sclerotiorum . Pyrrolnitrin, aminopyrrolnitrin, and monochloroaminopyrrolnitrin were isolated from J82 and identified by using thin-layer chromatography, high-performance liquid chromatography, and electron impact-mass, UV, and infrared spectroscopy . In growth chamber experiments, two antibiosis-negative mutants were not different from the parent strain in protecting the seeds from the fungus.

Clin Infect Dis, 1992 May, 14(5), 1078 - 83
Infections and pseudoinfections due to povidone-iodine solution contaminated with Pseudomonas cepacia; Panlilio AL et al.; In 1989 we investigated the first instance of Pseudomonas cepacia infections due to intrinsic contamination of a povidone-iodine product . Six patients in a Texas pediatric facility had P . cepacia infection or pseudoinfection (three, peritonitis; one, pseudoperitonitis; and two, pseudobacteremia) . Epidemiological studies showed one risk factor for infection of peritoneal fluid with P . cepacia: performance of peritoneal dialysis in the dialysis unit with use of one lot of povidone-iodine later found to be intrinsically contaminated (4/5 vs . 0/16, P = .001) . Blood cultures yielded P . cepacia after nurses wiped the tops of blood culture bottles with the povidone-iodine solution before inoculation . P . cepacia was cultured from three povidone-iodine containers used at the hospital and from four containers of the same lot obtained from other health-care facilities in Texas and California . Isolates from patients and the povidone-iodine had similar antibiograms, identical plasmid profiles, and identical DNA banding patterns on the basis of results of ribonucleotide typing . This investigation demonstrates that intrinsic contamination of povidone-iodine solution with P . cepacia can result in infections in addition to colonization and/or pseudoinfection.

Infect Immun, 1992 May, 60(5), 2002 - 7
Fimbriation of Pseudomonas cepacia; Kuehn M et al.; Fimbriae (pili) on the surface of bacteria have been suggested to facilitate adherence to mucosal epithelial surfaces . Three Pseudomonas cepacia cystic fibrosis isolates were screened for their ability to agglutinate erythrocytes (HA), a characteristic of some fimbrial types . One strain, designated PC103, was HA+, while another, PC109, was HA- . A fimbriated (f+) HA+ derivative of PC109 (PC2(13)) was selected by repeated erythrocyte adsorption . The two HA+ strains were shown by transmission electron microscopy to possess fimbriae which averaged 4.8 +/- 1.36 nm in width and 200 to greater than 2,100 nm in length (PCE2(13)) and 3.4 to 11.4 nm in diameter and 280 to 720 nm in length (PC103) . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins prepared from PC103, PC109, and PCE2(13) indicated that the putative fimbrial subunit had a mass of 16 kDa . Western blot (immunoblot) analysis of sheared cell supernatants indicated that the 16-kDa subunit from PC103 and PCE2(13) reacted with antibody to the P . aeruginosa PAK pilin subunit . Southern blot analysis of a SalI digest of PC103 DNA showed DNA fragments which hybridized to P . aeruginosa PAK probes containing either the pilin structural gene (pilA) or the pilin accessory genes (pilB, -C, and -D) but not the conserved N-terminal region of pilA . A 15-kb band was common to both hybridizations, indicating that this fragment contains the PC103 fimbrial gene cluster . These results indicated the presence of homology between P . aeruginosa PAK and PC103 fimbriae but also suggested that the P . cepacia fimbriae are not type IV-like . The importance of fimbriae in adherence to A549 cells (type II pneumocytes) was assessed with PC109 (f-) and PCE2(13) (f+) . PCE2(13) had an approximately 20-fold-higher level of adherence to A549 cells than PC109 . This suggested that fimbriation of P . cepacia is associated with increased adherence in vitro.

Somat Cell Mol Genet, 1992 May, 18(3), 227 - 31
A mutation in codon 717 of the CHO-K1 elongation factor 2 gene prevents the first step in the biosynthesis of diphthamide; Foley BT et al.; The histidine residue at position 715 of elongation factor 2 (EF-2) is posttranslationally modified in a series of enzymatic reactions to 2-{3-carboxyamido-3-(trimethylammonio)-propyl}histidine, which has been given the trivial name diphthamide . The diphthamide residue of EF-2 is the target site for ADP ribosylation by diphtheria toxin and Pseudomonas exotoxin A . ADP-ribosylated EF-2 does not function in protein synthesis . EF-2 that has not been posttranslationally modified at histidine 715 is resistant to ADP ribosylation by these toxins . In this report we show that a G-to-A transition in the first position of codon 717 of the EF-2 gene results in substitution of arginine for glycine and prevents addition of the side chain of diphthamide to histidine 715 of EF-2 . EF-2 produced by the mutant gene is fully functional in protein synthesis.

Mol Gen Genet, 1992 May, 233(1-2), 25 - 32
Construction of a yeast artificial chromosome library of tomato and identification of cloned segments linked to two disease resistance loci; Martin GB et al.; We have constructed a yeast artificial chromosome (YAC) library of tomato for chromosome walking that contains the equivalent of three haploid genomes (22,000 clones) . The source of high molecular weight DNA was leaf protoplasts from the tomato cultivars VFNT cherry and Rio Grande-PtoR, which together contain loci encoding resistance to six pathogens of tomato . Approximately 11,000 YACs have been screened with RFLP markers that cosegregate with Tm-2a and Pto - loci conferring resistance to tobacco mosaic virus and Pseudomonas syringae pv . tomato, respectively . Five YACs were identified that hybridized to the markers and are therefore starting points for chromosome walks to these genes . A subset of the library was characterized for the presence of various repetitive sequences and YACs were identified that carried TGRI, a repeat clustered near the telomeres of most tomato chromosomes, TGRII, an interspersed repeat, and TGRIII, a repeat that occurs primarily at centromeric sites . Evaluation of the library for organellar sequences revealed that approximately 10% of the clones contain chloroplast sequences . Many of these YAC clones appear to contain the entire 155 kb tomato chloroplast genome . The tomato cultivars used in the library construction, in addition to carrying various disease resistance genes, also contain the wild-type alleles corresponding to most recessive mutations that have been mapped by classical linkage analysis . Thus, in addition to its utility for physical mapping and genome studies, this library should be useful for chromosome walking to genes corresponding to virtually any phenotype that can be scored in a segregating population.

AJR Am J Roentgenol, 1992 May, 158(5), 1007 - 9
Treatment of bile duct stones by laser lithotripsy: results in 12 patients; Dawson SL et al.; We used a pulsed tunable dye laser (operating at 60 mJ per pulse, 504-nm wavelength) to fragment large (0.8-4.5 cm) stones retained in the hepatic ducts or common bile duct in 12 patients after cholecystectomy . Attempts to extract stones via a T-tube or endoscope had been unsuccessful in all patients . In nine of 12 patients, all stone fragments were successfully eliminated during the initial treatment . In one patient, fragmentation occurred but debris remained, requiring endoscopic stenting . Pseudomonas sepsis developed in this patient 30 days after the procedure and was treated by extraction of the stone fragments . Fragments remaining after lithotripsy were cleared at the same sitting by using saline flushing or endoscopic or percutaneous basket extraction . In two of 12 patients, the treatment was unsuccessful because of laser malfunction . The treatment was performed without complications, except for clinically insignificant hyperamylasemia, which occurred in two patients . Our experience suggests that laser lithotripsy offers a safe alternative for nonsurgical treatment of large retained biliary stones for patients in whom traditional treatments have failed.

J Bacteriol, 1992 May, 174(9), 3021 - 9
Regulation of tabtoxin production by the lemA gene in Pseudomonas syringae; Barta TM et al.; Pseudomonas syringae pv . coronafaciens, a pathogen of oats, was mutagenized with Tn5 to generate mutants defective in tabtoxin production . From a screen of 3,400 kanamycin-resistant transconjugants, seven independent mutants that do not produce tabtoxin (Tox-) were isolated . Although the Tn5 insertions within these seven mutants were linked, they were not located in the previously described tabtoxin biosynthetic region of P . syringae . Instead, all of the insertions were within the P . syringae pv . coronafaciens lemA gene . The lemA gene is required by strains of P . syringae pv . syringae for pathogenicity on bean plants (Phaseolus vulgaris) . In contrast to the phenotype of a P . syringae pv . syringae lemA mutant, the Tox- mutants of P . syringae pv . coronafaciens were still able to produce necrotic lesions on oat plants (Avena sativa), although without the chlorosis associated with tabtoxin production . Northern (RNA) hybridization experiments indicated that a functional lemA gene was required for the detection of a transcript produced from the tblA locus located in the tabtoxin biosynthetic region . Marker exchange mutagenesis of the tblA locus resulted in loss of tabtoxin production . Therefore, both the tblA and lemA genes are required for tabtoxin biosynthesis, and the regulation of tabtoxin production by lemA probably occurs at the transcriptional level.

J Neurosurg, 1992 May, 76(5), 838 - 44
In vitro efficacy of transferrin-toxin conjugates against glioblastoma multiforme; Hall WA et al.; The cytotoxic activity of immunotoxins constructed with human diferric transferrin (Tfn) as the carrier ligand and an abrin variant Pseudomonas exotoxin A (PE) and the diphtheria toxin mutant cross-reacting material (CRM) 107 as the toxin moieties were studied in vitro . Three malignant human cell lines, the glioblastomas multiforme SNB19 and SF295 and the LOX melanoma, and a nonhuman control murine melanoma cell line B16 were assessed . The presence of transferrin receptors on the cell lines was confirmed by direct 125I-Tfn binding assays . The 50% protein synthesis inhibitory concentration (IC50) values for all cell lines demonstrated that Tfn-abrin variant and Tfn-PE had comparable potency and were both more effective than Tfn-CRM 107 . Monensin, a carboxylic ionophore, potentiated the effect of Tfn-abrin variant against glioma cells approximately 35-fold with IC50 values of 4.0 x 10(-13) M and 4.7 x 10(-12) M for SNB19 and SF295, respectively . Cytotoxic activity of Tfn-abrin variant (with or without monensin) and Tfn-PE was correlated with the degree of Tfn receptor expression measured on the cell lines . The exquisite in vitro cytotoxicity of Tfn-abrin variant and Tfn-PE immunotoxins against glioma and melanoma cells warrants further in vivo evaluation and future consideration of these agents for potential clinical application against glioblastoma multiforme and leptomeningeal neoplasia.

Mol Gen Mikrobiol Virusol, 1992 May-Jun, (5-6), 13 - 6
{Mobilization using incompatibility group P1 plasmids in strains of Pseudomonas pseudomallei and Pseudomonas mallei as potential vectors for DNA cloning}; Anishchenko MA et al.; The cells of Pseudomonas pseudomallei and Pseudomonas mallei have been shown to serve as recipients for the plasmid RSF1010 and its recombinant derivatives pVA1 and pVA4 . The conjugative plasmids RP1 and pTH10 of the incompatibility group P1 are able to mobilize the nontransmissive vector plasmids for conjugation transfer into Pseudomonas pseudomallei and Pseudomonas mallei strains . The SmR determinant of the plasmid RSF1010 is expressed in the latter strains . These data makes the mentioned vector plasmids the candidates for DNA cloning in these strains.

J Chromatogr, 1992 Apr 24, 597(1-2), 155 - 66
Membrane-based receptor affinity chromatography; Nachman M et al.; Membrane-based receptor affinity chromatography (MRAC), which utilizes the molecular recognition between an immobilized receptor and its soluble protein ligand, has been developed for the purification of human interleukin-2 and related biomolecules . The multi-purpose affinity membrane used in this study consisted of a soluble form of interleukin-2 receptor (IL-2R) chemically bonded to hollow-fiber membranes in an oriented fashion . A model system involving anti-Tac-H (a humanized monoclonal antibody to IL-2R) was used to study the important factors influencing the performance of MRAC, including support morphology, mass transfer rate and adsorption kinetics . All three are shown to be highly efficient . MRAC has been successfully applied to the purification of anti-Tac-H, recombinant human interleukin-2 (rIL-2) and interleukin 2-Pseudomonas exotoxin fusion protein (IL2-PE40) . Overall, MRAC was found to be a viable, scalable and extremely productive affinity purification method.

Eur J Biochem, 1992 Apr 15, 205(2), 459 - 66
Characterization of 4-hydroxyphenylpyruvate dioxygenase . Primary structure of the Pseudomonas enzyme; Ruetschi U et al.; The primary structure of Pseudomonas 4-hydroxyphenylpyruvate dioxygenase was determined . Sequence degradation of the intact protein and of peptides from three different digests of the carboxymethylated protein established a 357-residue polypeptide chain with a free alpha-amino group . Hydroxylamine cleavage at a single Asn-Gly sequence was useful . Comparisons with known structures in data banks revealed no close relationship with other characterized proteins . The human enzyme has a related composition, suggesting that also the eukaryotic form belongs to this protein type, but with a blocked N-terminus like in many other eukaryotic intracellular proteins . Secondary structure predictions suggest an alpha/beta mixed structure, fairly typical of globular proteins, without long segments of hydrophobicity or charge, although a region in the middle of the C-terminal third of the subunit appears to have the most extreme properties . A ferric centre, correlating with enzyme activity and absorbance at 595 nm, has previously been assigned to tyrosinate coordination . The Tyr and His distributions, and the position of a single Cys residue, all suggest a few likely sites, outside the C-terminal segment, for this centre.

J Biol Chem, 1992 Apr 15, 267(11), 7224 - 39
Substrate specificity of Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase . Analysis of fatty acid analogs containing carbonyl groups, nitrogen heteroatoms, and nitrogen heterocycles in an in vitro enzyme assay and subsequent identification of inhibitors of human immunodeficiency virus I replication; Devadas B et al.; Covalent attachment of myristic acid (C14:0) to the amino-terminal glycine residue of a variety of eukaryotic cellular and viral proteins can have a profound influence on their biological properties . The enzyme that catalyzes this modification, myristoyl-CoA-protein N-myristoyltransferase (NMT), has been identified as a potential target for antiviral and antifungal therapy . Its reaction mechanism is ordered Bi Bi with myristoyl-CoA binding occurring before binding of peptide and CoA release preceding release of myristoylpeptide . Perturbations in the binding of its acyl-CoA substrate would therefore be expected to have an important influence on catalysis . We have synthesized 56 analogs of myristic acid (C14:0) to further characterize the acyl-CoA binding site of Saccharomyces cerevisiae NMT . The activity of fatty acid analogs was assessed using a coupled in vitro assay system that employed the reportedly nonspecific Pseudomonas acyl-CoA synthetase, purified S . cerevisiae NMT, and octapeptide substrates derived from residues 2-9 of the catalytic subunit of cyclic AMP-dependent protein kinase and the Pr55gag polyprotein precursor of human immunodeficiency virus I (HIV-I) . Analysis of ketocarbonyl-, ester-, and amide-containing myristic acid analogs (the latter in two isomeric arrangements, the acylamino acid (-CO-NH-) and the amide (-NH-CO)) indicated that the enzyme's binding site is able to accommodate a dipolar protrusion from C4 through C13 . This includes the region of the acyl chain occurring near C5-C6 (numbered from carboxyl) that appears to be bound in a bent conformation of 140-150 degrees . The activities of NMT's acyl-CoA substrates decrease with increasing polarity . This relationship was particularly apparent from an analysis of a series of analogs in which the hydrocarbon chain was terminated by (i) an azido group or (ii) one of three nitrogen heterocycles (imidazole, triazole, and tetrazole) alkylated at either nitrogen or carbon . This inverse relationship between polarity and activity was confirmed after comparison of the activities of the closely related ester- or amide-containing tetradecanoyl-CoA derivatives . Members from all of the analog series were surveyed to determine whether they could inhibit replication of human immunodeficiency virus I (HIV-I), a retrovirus that depends upon N-myristoylation of its Pr55gag for propagation . 12-Azidododecanoic acid was the most active analog tested, producing a 60-90% inhibition of viral production in both acutely and chronically infected T-lymphocyte cell lines at a concentration of 10-50 microM without associated cellular toxicity.

Nucleic Acids Res, 1992 Apr 11, 20(7), 1755 - 62
Upstream binding sequences of the XylR activator protein and integration host factor in the xylS gene promoter region of the Pseudomonas TOL plasmid; Holtel A et al.; The xylR and xylS genes, which encode the positive regulators of the TOL plasmid catabolic pathways, are adjacent genes on the TOL plasmid and are transcribed from divergent promoters . Transcription from the xylS gene promoter, Ps, is positively regulated by effector-activated XylR protein and requires the specific RNA polymerase sigma 54 subunit (RpoN) . Deletions and point mutations in the Ps upstream region localized the site of XylR interaction to the region between -133 bp and -207 bp (with respect to the transcriptional start of the xylS messenger), which contains an inverted sequence repeat largely homologous to the motif recognised by XylR in the XylR-regulated 'upper' catabolic pathway promoter, Pu . Gel retardation experiments showed binding of IHF to the Ps promoter region . Corresponding sequences showing good homology to the IHF-binding consensus were identified close to the Ps Promoter (between -35 bp and -47 bp, Ps proximal site) and further upstream overlapping the XylR recognition sequence (Ps distal site) . In the latter case IHF recognition motifs were found well conserved on both strands at nearly the same position (between -140 bp and -152 bp on the upper and between -141 bp and -153 bp on the lower strand) . Expression from Ps, either under inducing or non-inducing conditions, was, however, only slightly influenced by the absence of IHF in an IHF-deficient mutant and thus activation of Ps, like that of other sigma 54-dependent promoters which are rich in Ts, does not absolutely require IHF protein.

Biochim Biophys Acta, 1992 Apr 8, 1120(2), 208 - 14
Substrate specificity and kinetic properties of pepstatin-insensitive carboxyl proteinase from Pseudomonas sp . No . 101; Oda K et al.; The substrate specificity of the pepstatin-insensitive carboxyl proteinase isolated from Pseudomonas sp . No . 101 was studied by using a series of synthetic chromogenic substrates with general structure P5-P4-P3-P2-P1 *(NO2)Phe-Arg-Leu (P5, P4, P3, P2, P1: a variety of amino acids, (NO2)Phe is p-nitro-L-phenylalanine) . The nature of the residues occupying the P2, P3 and P4 positions as well as P1 position had strong influences on kinetic parameters . Among those tested, Lys-Pro-Ile-Glu-Phe*(NO2)Phe-Arg-Leu was the best substrate (Km = 3 microM, kcat = 6.9 s-1, kcat/Km = 2300 mM-1 s-1) . The S2 subsite of the enzyme was found to contain one or more basic amino acids while the S4 subsite probably includes one or more acidic amino acids . The pH-dependence of the hydrolysis of Ser-Pro-Ala-Lys-Phe*(NO2)Phe-Arg-Leu was studied . The pK1 and pK2 values for enzyme-substrate complex were found to be 2.97 and 4.92, respectively . Coupled with other results, it seems likely that two active carboxyl residues are involved in the catalytic action of the enzyme . In addition, it was found that a specific peptide inhibitor of the enzyme, tyrostatin, is a compeptive inhibitor with a ki value of 2.6 nM.

Invest New Drugs, 1992 Apr, 10(1), 17 - 22
Activity of a recombinant transforming growth factor-alpha-Pseudomonas exotoxin hybrid protein against primary human tumor colony-forming units; Von Hoff DD et al.; Transforming growth factor-alpha-Pseudomonas exotoxin-40 (TP40) is a recombinant fusion protein . TP40 consists of the entire human transforming growth factor-alpha (TGF alpha) protein fused to a 40,000 Da . segment of the Pseudomonas exotoxin A protein . TP40 is a bifunctional molecule that possesses the epidermal growth factor (EGF) receptor binding properties of TGF alpha and the cell killing properties of Pseudomonas exotoxin A . These properties make TP40 a selective cytotoxic agent that kills EGF receptor bearing cells . TP40 has been shown to effectively kill human tumor cell lines that possess EGF receptors in vitro and in nude mice . In the present study, TP40 was tested against tumors taken directly from patients and grown in a soft agar human tumor cloning system . A total of 107 patients' tumors (taken from patients with tumors refractory to chemotherapy) were tested with a continuous exposure to 0.5-50 nM concentrations of the agent . TP40 exhibited a clear dose response effect against a wide variety of human solid tumor colony-forming units with greater than or equal to 84% of evaluable tumors responding at a drug concentration greater than or equal to 24 nM . When used as a continuous exposure, concentrations of TP40 as low as 5 nM demonstrated substantial in vitro activity . This activity included cytotoxicity against breast, colorectal, endometrial, head and neck, non small-cell lung, gastric, sarcoma, and pancreatic cancer tumor colony-forming units . Additional in vivo testing of this compound is warranted.

Appl Environ Microbiol, 1992 Apr, 58(4), 1276 - 83
Isolation of Pseudomonas pickettii strains that degrade 2,4,6-trichlorophenol and their dechlorination of chlorophenols; Kiyohara H et al.; Three strains of Pseudomonas pickettii that can grow with 2,4,6-trichlorophenol (2,4,6-TCP) as the sole source of carbon and energy were isolated from different mixed cultures of soil bacterial populations that had been acclimatized to 2,4,6-TCP . These strains released 3 mol of chloride ion from 1 mol of 2,4,6-TCP during the complete degradation of the TCP . Of these strains, P . pickettii DTP0602 in high-cell-density suspension cultures dechlorinated various chlorophenols (CPs) . Cells that were preincubated with 2,4,6-TCP converted isomers of 4-CP to the corresponding chloro-p-hydroquinones, but those preincubated with 4-CP converted CPs lacking a chlorine atom(s) at the o position to isomers of chlorocatechol . The ability of DTP0602 to dechlorinate 2,4,6-TCP was induced by 2,6-dichlorophenol, 2,3,6- and 2,4,6-TCP, and 2,3,4,6-tetrachlorophenol and was repressed in the presence of succinate or glucose.

Arch Dis Child, 1992 Apr, 67(4), 479 - 81
Raised serum soluble interleukin-2 receptor concentrations in cystic fibrosis patients with and without evidence of lung disease; Dagli E et al.; Soluble interleukin-2 receptor (sIL-2R-CD25) concentrations were measured in the sera of 115 children with cystic fibrosis and 45 aged matched controls . Above the age of 4 years children with cystic fibrosis had significantly raised concentrations irrespective of disease status as judged by Shwachman score, lung function, or evidence of pseudomonas colonisation . It is believed that these data indicate that T lymphocyte activation can be detected before there is clinical evidence of lung inflammation due to infection in cystic fibrosis . They support the notion that early use of anti-inflammatory (immunosuppressive) drugs may have a role in delaying the progress of lung damage in cystic fibrosis.

Blood, 1992 Apr 1, 79(7), 1775 - 80
Interleukin-6 fused to a mutant form of Pseudomonas exotoxin kills malignant cells from patients with multiple myeloma; Kreitman RJ et al.; IL-6-PE4E is a recombinant protein consisting of interleukin-6 (IL-6) fused to a mutant form of Pseudomonas exotoxin in which four basic amino acids are changed to glutamate (PE4E) . The chimeric toxin has been previously shown to specifically kill malignant hepatic, prostatic, epidermoid, and myeloma cell lines in vitro . To explore the possible clinical utility of IL-6-PE4E, particularly as an agent for ex vivo purging of marrow for autologous bone marrow transplantation (ABMT), we tested malignant cells from patients with multiple myeloma for sensitivity to this chimeric toxin . Ficoll-purified bone marrow cells were incubated with and without IL-6-toxin for 2 to 3 days . Eight of the 15 myeloma patients had cells that were sensitive to IL-6-toxin as measured by a decrease in the level of protein synthesis . Cells from five patients were very sensitive to IL-6-PE4E, with 50% inhibition of protein synthesis (ID50) achieved at or below 6 ng/mL (7 x 10(-11) mol/L) . Cells from three additional patients showed moderate sensitivity, with ID50s between 30 and 140 ng/mL . The remaining seven samples showed little or no sensitivity, with ID50s greater than or equal to 400 ng/mL . Normal bone marrow cells or normal BFU-E and CFU-GM were resistant to the IL-6-toxin even at 1,000 ng/mL . Neither IL-6, IL-2-PE4E, nor an enzymatically deficient mutant of IL-6-PE4E was cytotoxic toward the myeloma cells, indicating that the cytotoxic effect of IL-6-PE4E required the adenosine diphosphate-ribosylation function as well as the specific ligand . Our data suggest that IL-6-toxin could be effective in ex vivo marrow purging in selected multiple myeloma patients who are candidates for ABMT, and that this toxin should also be investigated further for in vivo therapy.

Proc Natl Acad Sci U S A, 1992 Apr 1, 89(7), 3075 - 9
Independent domain folding of Pseudomonas exotoxin and single-chain immunotoxins: influence of interdomain connections; Brinkmann U et al.; We have studied the refolding of completely unfolded and reduced Pseudomonas exotoxin (PE) and of recombinant single-chain immunotoxins made with monoclonal antibody B3 that are composed of a heavy-chain variable region connected by a flexible linker to the corresponding light-chain variable region (Fv), which is in turn fused to a truncated form of PE . We have found by direct activity assays that different functional domains of these multifunctional proteins fold independently with different kinetics . The ADP-ribosylation domain of PE and of the recombinant immunotoxin fold rapidly, whereas the assembly of the binding and/or translocation domains is regained more slowly . The complete refolding of native PE occurs more rapidly than the refolding of the recombinant immunotoxins . To determine the influence of the connector region between the B3(Fv) moiety and the toxin on the folding process of the recombinant immunotoxin B3(Fv)-PE38KDEL, we have made two different mutations in the peptide that connects the single-chain Fv domain to domain II of PE . These molecules show different folding kinetics, differences in their propensity to aggregate, and different yields of correctly folded molecules . A mutation that decreases aggregation increases the rate of formation and the yield of active immunotoxin molecules.

Proc Natl Acad Sci U S A, 1992 Apr 1, 89(7), 3065 - 9
Alteration of a protease-sensitive region of Pseudomonas exotoxin prolongs its survival in the circulation of mice; Brinkmann U et al.; Pseudomonas exotoxin A (PE) is a single-chain 66-kDa polypeptide that kills eukaryotic cells by ADP-ribosylation of translational elongation factor 2 . PE is composed of three major structural domains whose functions are binding of cells (I), translocation (II), and ADP-ribosylation (III) . Here we describe a protease cleavage target that is located near arginine-490 on the surface of domain III . We made several different types of mutations near arginine-490 . Deletion of arginine-490 or replacement of arginine-490 and -492 with serine and lysine or with two lysines resulted in protease-resistant molecules that were fully cytotoxic and had normal ADP-ribosylation activity . However, the half-life in mouse blood of the PE delta 490 mutant was 24 min whereas that of PE was 13 min . Furthermore, two PE mutants that were protease-hypersensitive, PEGlu246,247,249 and PEGlu57,246,247,249 (in which glutamate residues replace basic residues at the indicated positions), had very short half-lives . These data indicate that protease sensitivity is an important determinant in the half-life of PE in the circulation and suggest that the half-life of other proteins may be prolonged by removal of protease sites . Deletion of arginine-492 or the replacement of amino acids 486-491 with three glycines markedly diminished ADP-ribosylation activity and cytotoxicity, indicating that this region of domain III is also important for catalytic activity.

J Bacteriol, 1992 Apr, 174(7), 2323 - 31
Structural and biochemical characterization of the Escherichia coli argE gene product; Meinnel T et al.; The DNA sequence of a 2,100-bp region containing the argE gene from Escherichia coli has been determined . The nucleotide sequence of the ppc-argE intergenic region was also solved and shown to contain six tandemly repeated REP sequences . Moreover, the oxyR gene has been mapped on the E . coli chromosome and shown to flank the arg operon . The codon responsible for the translation start of argE was determined by using site-directed mutants . This gene spans 1,400 bp and encodes a 42,350-Da polypeptide . The argE3 allele and a widely used argE amber gene have also been cloned and sequenced . N-Acetylornithinase, the argE product, has been overproduced and purified to homogeneity . Its main biochemical and catalytic properties are described . Moreover, we demonstrate that the protein is composed of two identical subunits . Finally, the amino acid sequence of N-acetylornithinase is shown to display a high degree of identity with those of the succinyldiaminopimelate desuccinylase from E . coli and carboxypeptidase G2 from a Pseudomonas sp . It is proposed that this carboxypeptidase might be responsible for the acetylornithinase-related activity found in the Pseudomonas sp.

Chem Phys Lipids, 1992 Apr, 61(2), 193 - 8
Stereoselectivity of lipases: esterification reactions of octadecylglycerol; Meusel D et al.; Stereoselectivity of several triacylglycerol lipases (EC 3.1.1.3) has been investigated in the enzymatic esterification of rac-1-O-octadecylglycerol with oleic acid in the presence of organic solvents, such as hexane . X-1(3)-O-Octadecylmonooleoylglycerols were the only products formed with most lipases; considerable proportions of X-1(3)-O-octadecyldioleoylglycerols were also formed with the lipase from Candida cylindracea . The mixtures of unesterified enantiomeric substrates, i.e., X-1(3)-O-octadecylglycerols were converted to their 3,5-dinitrophenylurethane derivatives and subsequently resolved into sn-1 and sn-3 enantiomers by HPLC on a chiral stationary phase (Sumichiral OA 2100) . The data on enantiomeric excess (ee) and enantiomeric ratio (E) in the unesterified substrate revealed for the lipases from porcine pancreas, Rhizopus sp., Pseudomonas sp., Candida cylindracea, Chromobacterium viscosum and Penicillium cyclopium a distinct preference for 1-O-octadecyl-sn-glycerol over its enantiomer indicating stereoselectivity for the sn-3 position . For the lipase from Rhizomucor miehei a slight stereoselectivity for the sn-1 position was observed . Solvents, such as diethyl ether and dichloromethane, strongly inhibited the esterification reaction, but the enzymatic activity could be restored upon removal of such solvents by washing with hexane indicating reversible inhibition.

Appl Biochem Biotechnol, 1992 Apr, 33(1), 15 - 24
Enzymatic synthesis of (R) and (S) 1-deuterohexanol; Bradshaw CW et al.; This paper describes practical enzymatic procedures for the synthesis of (R) and (S) 1-deuterohexanol, a useful building block for chiral poly isocyanated liquid crystals . Alcohol dehydrogenases from horse liver and Pseudomonas catalyzed the reduction of hexanal with deuterated NAD (NADD) resulting in 50% and 89% yields of (R) and (S) 1-deuterohexanol, respectively . The deuterated cofactor was regenerated in situ by alcohol dehydrogenase catalyzed oxidation of ethanol-d6 or 2-propanol-d8 . The (S) alcohol was also synthesized by the horse liver alcohol dehydrogenase reduction of 1-deuterohexanal, which was prepared chemically from hexanal . The yields of the reaction were greatly increased by the use of a biphasic system or with the immobilized enzyme in anhydrous organic solvents . Horse liver alcohol dehydrogenase was stabilized by immobilization on PAN or noncovalent entrapment on XAD resin.

Can J Microbiol, 1992 Apr, 38(4), 309 - 12
Transposon Tn5-259 mutagenesis of Pseudomonas cepacia to isolate mutants deficient in antifungal activity; Jayaswal RK et al.; Transposon Tn5-259 was inserted into the chromosome of Pseudomonas cepacia by mating with an Escherichia coli strain harboring a self-mobilizable, temperature-sensitive plasmid, pME12 . Data from Southern blots and auxotroph analyses indicated that a single copy of the transposon was inserted in several places into the chromosome of P . cepacia . Among 1500 Tn5-259 transconjugants, only one mutant was found to be defective in the production of an antifungal compound, pyrrolnitrin . In addition, this mutant lost its ability to antagonize fungal phytopathogens . Using flanking DNA of the mutated gene as a probe, we have isolated four overlapping cosmid clones from a genomic library of P . cepacia . However, we were unable to complement the mutant because of difficulty in mobilizing the cosmids from E . coli to P . cepacia.

Infect Immun, 1992 Apr, 60(4), 1434 - 40
Identification of the mucin-binding adhesin of Pseudomonas cepacia isolated from patients with cystic fibrosis; Sajjan SU et al.; In previous experiments, we have shown that isolates of Pseudomonas cepacia from sputa of patients with cystic fibrosis (CF), particularly those with severe lung infection, exhibited specific binding to purified respiratory or intestinal mucins (U . Sajjan, M . Corey, M . Karmali, and J . Forstner, J . Clin . Invest . 89:648-656, 1992) . The present report describes the identification of the adhesin as a protein located on fimbriae of mucin-binding P . cepacia . From a total of 53 isolates available (from 22 patients with CF), we used three mucin-binding and three non-mucin-binding isolates for our experiments . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude P . cepacia homogenates was performed, the separated proteins were blotted onto nitrocellulose and overlaid with purified mucin, and mucin-binding components were detected with an antimucin antibody and then a second-antibody-alkaline phosphatase conjugate system . Only mucin-binding isolates exhibited a positively stained band at an Mr of 22,000 . The 22-kDa protein was purified, and a polyclonal antibody specific for it was developed in rabbits . By electron microscopy and immunogold labelling, both the antibody and mucin (separately) were localized to pili present over the entire surface of the bacterial cells . Non-mucin-binding isolates did not have (or had very few) pili and did not stain with either mucin or the antibody to the 22-kDa protein . The purified 22-kDa protein and its antibody were each able to inhibit piliated P . cepacia binding to mucin . The amino acid composition of the 22-kDa protein was dissimilar to those of the major pilin proteins of Escherichia coli (type 1 pilus) and P . aeruginosa (PAK and PAO1 strains) . Both the pili of P . aeruginosa PAK and PAO1 and antibodies to these pili failed to inhibit P . cepacia binding to mucin . Thus, P . cepacia adhesion to mucin is mediated by a pilin-associated 22-kDa protein which differs from epithelial-cell-binding pilin proteins of P . aeruginosa . We postulate that the 22-kDa adhesin may play a role in the virulence of P . cepacia lung infections of patients with CF.

J Gen Microbiol, 1992 Apr, 138 ( Pt 4), 701 - 8
Molecular analysis of an esterase-encoding gene from a lipolytic psychrotrophic pseudomonad; McKay DB et al.; An esterase gene (estA) from a lipolytic psychotroph (Pseudomonas sp . LS107d2), was cloned in Escherichia coli and its nucleotide sequence was determined, revealing an ORF encoding a polypeptide of 389 amino acid residues, with a molecular mass of 42276 Da . Labelling of plasmid-encoded proteins with {35S}methionine, using the maxicell procedure, gave a single polypeptide of molecular mass 42 kDa, consistent with that calculated from the ORF . Colonies of E . coli cells containing estA produced a clear halo when grown on solid media containing tributyrin; no clearance was produced when cells were grown on media containing triolein . Extracts of cells containing estA also hydrolysed water-soluble nitrophenol esters, but were unable to cleave water-insoluble substrates . The preference for water-soluble substrates indicates that the gene product is an esterase.

Int J Immunopharmacol, 1992 Apr, 14(3), 465 - 72
Targeting growth factor receptors with fusion toxins; Kreitman RJ et al.; Recombinant toxins which bind to growth factor receptors have been prepared and used to kill cells responsible for malignant or autoimmune disease . Our strategy has been to genetically fuse ligands to different forms of Pseudomonas exotoxin which due to mutations or deletions do not bind to normal cells . The resulting recombinant chimeric toxins, in concentrations often less than 1 ng/ml, selectively kill cells expressing the appropriate growth factor receptor . The ligand may be a growth factor, such as transforming growth factor alpha (TGF alpha), interleukin 6 (IL6) or interleukin 2 (IL2), or single chain antigen binding proteins, such as the variable heavy and light regions of the monoclonal antibody anti-Tac . These chimeric toxins kill not only established cell lines but also fresh tumor cells from patients and display anti-tumor activity toward human malignant tumors in nude mice . While clinical trials are beginning with some of these agents, work continues to improve the effectiveness of recombinant chimeric toxins, and to widen the scope of disorders which might be treated by this approach.

Eur J Biochem, 1992 Apr 1, 205(1), 25 - 31
Characterization of the endogenous ADP-ribosylation of wild-type and mutant elongation factor 2 in eukaryotic cells; Fendrick JL et al.; Anti-{ADP-ribosylated elongation factor 2 (EF-2)} antiserum has been used to immunoprecipitate the modified form of EF-2 from polyoma-virus-transformed baby hamster kidney (pyBHK) cells {Fendrick, J . L . & Iglewski, W . J . (1989) Proc . Natl Acad . Sci . USA 86, 554-557} . This antiserum also immunoprecipitates a 32P-labelled protein of similar size to EF-2 from a variety of primary and continuous cell lines derived from many species of animals . One of these cell lines, chinese hamster ovary CHO-K1 cells was further characterized . The time course of labelling of ADP-ribosylated EF-2 with {32P}orthophosphate was similar in pyBHK cells and in CHO-K1 cells . The kinetics of labelling were more rapid for cells cultured in 2% serum than 10% serum, with incorporation of 32P reaching a maximum at 6 h and 10 h, respectively . EF-2 mutants of pyBHK and CHO-K1 cells resistant to diphtheria-toxin-catalyzed ADP-ribosylation of EF-2 remain sensitive to cellular ADP-ribosylation of EF-2 . The 32P-labelled moiety of ADP-ribosylated EF-2 was digested by snake venom phosphodiesterase and the product was identified as AMP . The same 32P-labelled tryptic peptide was modified by toxin in wild-type EF-2 and by the cellular transferase in mutant EF-2 . When purified EF-2 from pyBHK cells was incubated with {carbonyl-14C}nicotinamide and diphtheria toxin fragment A, under conditions for reversal of the ADP-ribosylation reaction, {14C}NAD was generated . The results suggest that cellular ADP-ribosylated EF-2 exists in a variety of cell types, and the ribosylated product is identical to that produced by toxin ADP-ribosylation of EF-2, except in diphthamide mutant cells . Studies with the mutant cell lines indicate that the toxin and the cellular transferase, however, recognize different determinants at the ADP-ribose acceptor site in EF-2 . The cellular transferase does not require the diphthamide modification of the histidine ring in the amino acid sequence of EF-2 for the transfer of ADP-ribose to the ring . Therefore, we would expect the cellular transferase active site to be similar to, but not identical to, the critical amino acids demonstrated in the active site of diphtheria toxin and Pseudomonas exotoxin A.

Biochem J, 1992 Mar 15, 282 ( Pt 3), 675 - 80
Bacterial metabolism of 5-aminosalicylic acid . Initial ring cleavage; Stolz A et al.; The metabolism of 5-aminosalicylate (5AS) by a bacterial strain, Pseudomonas sp . BN9, was studied . Intact cells of Pseudomonas sp . BN9 grown with 5AS oxidized 5AS and 2,5-dihydroxybenzoate (gentisate), whereas cells grown with gentisate oxidized only the growth substrate of all substituted salicylates tested . Cell extracts from Pseudomonas sp . BN9 catalysed the stoichiometric reaction of 1 mol of oxygen with 1 mol of 5AS to a metabolite with an intense u.v.-absorption maximum at 352 nm (pH 8.0) . This metabolite was accumulated under neutral conditions, but was rapidly destroyed at acid pH . It was identified by m.s . and acid-catalysed deamination to fumarylpyruvate (trans-2,4-dioxohept-5-enedioic acid) as cis-4-amino-6-carboxy-2-oxohexa-3,5-dienoate, thus demonstrating direct cleavage of the monohydroxylated substrate 5AS to a non-aromatic ring-fission product . The enzyme responsible for conversion of 5AS was shown to be Fe(II)-dependent and to be distinct from gentisate 1,2-dioxygenase in strain BN9.

Infect Immun, 1992 Mar, 60(3), 735 - 41
Inhibition of Aspergillus fumigatus elastase with monoclonal antibodies produced by using denatured elastase as an immunogen; Frosco M et al.; In preparing monoclonal antibodies to the elastase from Aspergillus fumigatus, we found that the enzyme was weakly immunogenic in BALB/c mice . Antiserum titers were only 1:1,000 to 1:5,000, and hybridomas secreted nonspecific immunoglobulin M (IgM) . Denaturing the elastase in 0.5% sodium dodecyl sulfate at 80 degrees C for 10 min prior to injection increased titers of antiserum against the nondenatured (native) enzyme 10-fold . Of eight hybridomas selected following immunization with the denatured enzyme, seven produced IgG reactive with the native enzyme and one produced nonspecific IgM . The nondenatured immunogen tested again yielded mainly IgM producers . Immunoblots and enzyme-linked immunosorbent assay showed that the IgG monoclonal antibodies were reactive with both the denatured and nondenatured fungal elastases; none cross-reacted with human neutrophil elastase, porcine pancreatic elastase, or Pseudomonas elastase . Elastase-specific polyclonal antibody produced in mice inhibited elastase activity beginning at a molar ratio (antibody to elastase) of 4:1, and activity was completely inhibited at 14.5:1 . Some individual monoclonal antibodies partially inhibited elastase, but certain pairs, at a molar ratio of each antibody to elastase of 5.4:1, acted synergistically to inhibit the activity completely.

Clin Pediatr (Phila), 1992 Mar, 31(3), 161 - 7
Impact of CF summer camp; Kaplan TA et al.; In two consecutive years, patients with cystic fibrosis were studied at the beginning and end of a nine-day summer camp program to assess the program's effects on weight gain and pulmonary function . The camp experience includes daily exercise and a high-protein and high-fat diet . There were a total of 58 children between 6 and 12 years of age (42 different patients) and 10 adult counselors from 19 to 30 years of age (eight different patients) . On the first and eighth days patients were weighed, sputum cultures were collected, and spirometry was performed . In year 2, peak expiratory flow rate was monitored daily . Also in year 2, campers and counselors with CF were prescreened by sputum culture and excluded from camp if they had Pseudomonas cepacia in their sputum . Only one candidate screened was positive before camp . In year 1, no significant group changes in pulmonary function were identified . In year 2, significant increases on post-camp testing were found for FEF 25%-75% and PEF . Mean body weight for all patients increased significantly, by 0.4 kg in year 1 and 0.9 kg in year 2 (p less than .05) . In year 1, a total of nine patients acquired a new organism in their follow-up sputum culture, including five who acquired a new Pseudomonas species . There was no intra-cabin pattern of spread . Four patients were positive for P . cepacia on day 1 culture . No new subjects acquired this organism on follow-up examination . In year 2, only one subject had P . cepacia on the first camp collection; he alone was positive on day 9.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1992 Mar 5, 224(1), 281 - 2
Crystallization and preliminary X-ray study of a lipase from Pseudomonas glumae; Cleasby A et al.; Lipase from Pseudomonas glumae has been purified and crystallized in two forms, using the hanging drop method of vapour diffusion at 4 degrees C and 15 degrees C . Both forms grew at pH 9.0 from 0.1 M-Tris buffer in the presence of 10% (v/v) acetone . Form 1 was crystallized from 27 to 29% polyethylene glycol in the presence of less than 0.5% (v/v) n-dodecyl-beta-D-glucopyranoside . Form 2 was grown from 17 to 19% ammonium sulphate in the presence of 1% n-octyl-beta-D-glucopyranoside . Form 1 is orthorhombic with space group P2(1)2(1)2(1), and cell dimensions of a = 158.1 A, b = 158.6 A, c = 63.4 A, Form 2 is tetragonal with space group P4(1)2(1)2 (or P4(3)2(1)2) and cell dimensions of a = 89.3 A, c = 180.4 A . Form 1 probably has four molecules per asymmetric unit and diffracts to at least 2.5 A . Form 2 has two molecules per asymmetric unit and diffracts to at least 3.0 A.

J Biol Chem, 1992 Mar 5, 267(7), 4844 - 53
Analysis of bph operon from the polychlorinated biphenyl-degrading strain of Pseudomonas pseudoalcaligenes KF707; Taira K et al.; The entire nucleotide sequences (6.8 kilobase pairs) of the bphABC genes and their products involved in the initial dioxygenation and ring-meta-cleavage of biphenyls and polychlorinated biphenyls were determined . The first bphA gene starts about a 100 base pairs downstream from the transcriptional initiation site . The bphA region, which encodes a cluster of enzymes including biphenyl dioxygenase catalyzing the initial catabolic step, consists of five open reading frames (ORFs) . Five proteins corresponding to these ORFs in the molecular masses were detected by in vitro protein synthesis, of which four ORFs are very similar to the recently reported todC1C2BA genes coding for the corresponding enzymes catalyzing the initial dioxygenation reactions of toluene (Zylstra, G.J., and Gibson, D . T . (1989) J . Biol . Chem . 264, 14940-14946) . The third open reading frame (ORF3) of the bphA region, missing its counterpart in the toluene dioxygenase gene cluster, was site-specifically deleted, and the resulting enzymatically active mutant reveals that this ORF3 is not mandatory for the catabolism of biphenyls . Thus the biphenyl degradation pathway and the responsible enzymes/genes are very similar to those of toluene degradation despite their discrete substrate specificity.

Mol Plant Microbe Interact, 1992 Mar-Apr, 5(2), 187 - 93
Transcriptional organization and expression of the large hrp gene cluster of Pseudomonas solanacearum; Arlat M et al.; Cloning and localized mutagenesis of the larger cluster of hrp genes of Pseudomonas solanacearum strain GMI1000 allowed the definition of the borders of this cluster, which now extends about 2 kb to the left of the insert of the previously described plasmid pVir2 (Boucher et al . 1987, J . Bacteriol . 169:5626-5632) . The size of the cluster has also been expanded 3 kb to the right to include a region previously described as dsp; our present data demonstrate that insertions occurring in these 3 kb lead to leaky mutations affecting both pathogenicity on tomato and ability to induce the hypersensitive response (HR) on tobacco . Therefore, the size of the entire hrp gene cluster is estimated to be about 22 kb . The use of transposon Tn5-B20, which promotes transcriptional gene fusions, allowed us to demonstrate that the hrp gene cluster is organized in a minimum of six transcriptional units, which are transcribed when the culture is grown in minimal medium but are repressed during growth in rich medium or in the presence of peptone or Casamino Acids . The level of expression in minimal medium is modulated by the carbon source provided; pyruvate is the best inducer . Under these conditions the level of expression observed in vitro appears to be representative of the actual expression observed in planta.

FEMS Microbiol Lett, 1992 Mar 1, 70(2), 147 - 51
Bacterial uptake of octyl ethanolamine increases with pH; Sandin M et al.; The uptake of octyl ethanolamine (C8EA) by Pseudomonas pseudoalcaligenes was determined at pH 7.1-10.0 . At pH 9.1 the total uptake was nearly three times higher and at pH 10.0 four times higher than at pH 7.1 . Also the initial rate of uptake was lowest at pH 7.1 . At pH 7.1 five to ten times higher concentrations of C8EA were needed than at pH 9.1 to achieve the same degree of leakage of cytoplasmic constituents . The results support the hypothesis that penetration of the bacterial cytoplasmic membrane by C8EA in its uncharged form is favoured . This takes place particularly with high pH in the suspending medium . In the cytoplasm, the pH is lower, and C8EA becomes more protonated . This will prevent back diffusion, promote accumulation and enhance membrane interaction and toxicity at high pH.

Mutagenesis, 1992 Mar, 7(2), 125 - 35
Resistance to Pseudomonas toxin A: a sensitive marker to screen for mutagenic substances using V79 cells; Suter W et al.; The goal of this study was the development of a mutagenicity assay in V79 cells using Pseudomonas toxin A (PTA) as a selective agent and to compare it with the V79/hypoxanthineguaninephosphoribosyl transferase (HGPT) assay . PTA inhibits protein synthesis by covalently modifying elongation factor 2 (EF-2) . Mutations in the EF-2 gene, in genes responsible for the modification of EF-2, and also in genes for the transport of the toxin into the cells, lead to PTA resistance . This involvement of several genes might explain the relatively high spontaneous mean mutation frequency of (122.7 +/- 38.8) x 10(-6) in growing cells (2 micrograms PTA/ml) as compared with (3.97 +/- 4.68) x 10(-6) in the HGPRT test (10 micrograms thioguanine/ml), where only mutations in the HGPRT gene lead to resistance . Methyl methanesulphonate, methyl nitroso urea and UV light were directly mutagenic in the V79/PTA assay . Aflatoxin B1 was mutagenic in the presence of an S9 mix . Benzidine, procarbazine, 2-acetylaminofluorene (2-AAF) and 4-acetylaminofluorene (4-AAF) were positive in the V79/PTA test when rat hepatocytes were used for metabolic activation . Using cells treated together with those used for the PTA assay, benzidine, 2-AAF and 4-AAF were negative in the V79/HGPRT test, while procarbazine was found to be positive under these test conditions . Hexamethyl phosphoramide (HMPA) was tested in the presence of rat hepatocytes and was negative in both test systems . Phorbol-12-myristate-13-acetate was highly cytotoxic, but did not induce mutations at the PTA resistance loci . In conclusion, it was demonstrated that the V79/PTA assay is an easy and sensitive method to screen for gene mutations in mammalian cells.

Cutis, 1992 Mar, 49(3), 185 - 6
Pseudomonas toe web infections; Westmoreland TA et al.; We present the case of a patient with a presumed tinea pedis infection who was referred to us after being treated with oral griseofulvin for two weeks with no improvement . We used a simple Wood's light examination and culture to establish the diagnosis of Pseudomonas toe web infection and achieved rapid resolution with oral ciprofloxacin treatment and local application of Castellani's paint.

J Bacteriol, 1992 Mar, 174(6), 1837 - 43
Physical and functional characterization of the gene cluster encoding the polyketide phytotoxin coronatine in Pseudomonas syringae pv . glycinea; Young SA et al.; Pseudomonas syringae pv . glycinea PG4180 produces the polyketide phytotoxin coronatine . The coronatine synthesis genes in PG4180 were previously shown to reside on a 90-kb plasmid designated p4180A . In the present study, clones containing a 34-kb region of p4180A were saturated with Tn5, and 71 unique mutations were recombined into p4180A by marker exchange . The effect of each mutation on coronatine synthesis was determined by analyzing the organic acids produced by the mutants by reverse-phase high-performance liquid chromatography . The organic acids of selected mutants were derivatized to their methyl esters and analyzed by gas chromatography and gas chromatography-mass spectrometry . Mutations in a 20.5-kb region of p4180A completely blocked the synthesis of coronafacic acid and coronatine . Mutations within a 4.4-kb region of p4180A prevented the formation of coronatine but allowed for production of coronafacic acid, coronafacoylvaline, coronafacoylisoleucine, and coronafacoylalloisoleucine . The phenotypes of selected mutants were further confirmed in feeding experiments in which coronafacic acid or coronamic acid was added to the culture media . The results of this study allow us to speculate on the likely sequence of steps in the later stages of coronatine biosynthesis.

J Bacteriol, 1992 Mar, 174(6), 1734 - 41
Organization and environmental regulation of the Pseudomonas syringae pv . syringae 61 hrp cluster; Xiao Y et al.; The ability of Pseudomonas syringae pv . syringae 61 to elicit the hypersensitive response in nonhost plant species has been linked to a cluster of hrp/hrm genes whose expression appears to be environmentally regulated . To understand the genetic organization of this hrp/hrm gene cluster and its expression during the interaction with nonhost plant species better, we constructed a set of chromosomal hrp-uidA fusions in P . syringae pv . syringae 61 by Tn5-gusA1 mutagenesis of the cloned hrp/hrm gene cluster and transferred them into the genome by marker exchange mutagenesis . Complementation analysis employing plasmid-borne Tn5-gusA1 insertions and previously characterized chromosomal TnphoA mutations defined at least eight apparent transcriptional units within the hrp/hrm cluster, several of which were multicistronic . The expression of hrp-uidA fusions in seven of these apparent hrp transcriptional units increased following inoculation into tobacco leaves . Enhanced expression from a representative fusion was detected 1 h after inoculation of tobacco leaves . The induction observed in planta was similar to the levels detected following culture of the bacteria in minimal-salts medium: irrespective of the carbon source . Complex amino acid sources, such as peptone, repressed the expression of P . syringae pv . syringae 61 hrp genes at levels exceeding 0.028% . The results indicate that enhanced expression of hrp genes occurs early in the interaction with nonhost plant species in an apparent response to altered nutritional conditions.

J Bacteriol, 1992 Mar, 174(5), 1604 - 11
The cloned avirulence gene avrPto induces disease resistance in tomato cultivars containing the Pto resistance gene; Ronald PC et al.; Resistance of tomato plants to the bacterial pathogen Pseudomonas syringae pv . tomato race 0 is controlled by the locus Pto . A bacterial avirulence gene was cloned by constructing a cosmid library from an avirulent P . syringae pv . tomato race, conjugating the recombinants into a strain of P . syringae pv . maculicola virulent on a tomato cultivar containing Pto, and screening for those clones that converted the normally virulent phenotype to avirulence . The cloned gene, designated avrPto, reduced multiplication of P . syringae pv . tomato transconjugants specifically on Pto tomato lines, as demonstrated by bacterial growth curve analyses . Analysis of F2 populations revealed cosegregation of resistance to P . syringae pv . tomato transconjugants carrying avrPto with resistance to P . syringae pv . tomato race 0 . Surprisingly, mutation of avrPto in P . syringae pv . tomato race 0 does not eliminate the avirulent phenotype of race 0, suggesting that additional, as yet uncharacterized, avirulence genes and/or resistance genes may contribute to specificity in the avrPto-Pto interaction . Genetic analysis indicates that this resistance gene(s) would be tightly linked to Pto . Interestingly, P . syringae pv . glycinea transconjugants carrying avrPto elicit a typical hypersensitive resistant response in the soybean cultivar Centennial, suggesting conservation of Pto function between two crop plants, tomato and soybean.

Mol Gen Mikrobiol Virusol, 1992 Mar-Apr, (3-4), 17 - 20
{Transformation of pathogenic pseudomonas by plasmid DNA}; Abaev IV et al.; The possibility has been shown of the genetical transformation of Pseudomonas mallei strains by the purified DNA of the plasmids RSF1010, pES154, pBS222 and pBR325 . The frequency of transformation varied from 1.2 x 10(1) to 2.0 x 10(2) depending on the plasmid DNA and transformation technique used in the experiments . Pseudomonas pseudomallei cells could not be transformed by the methods described in the paper.

J Med Microbiol, 1992 Mar, 36(3), 184 - 9
Isolation of a novel siderophore from Pseudomonas cepacia; Sokol PA et al.; A novel iron-binding compound was identified in ethyl acetate extracts of the supernates from Pseudomonas cepacia cultures . This compound, named azurechelin, was produced by 88% of P . cepacia strains isolated from the respiratory tract . Production of azurechelin was regulated by the iron concentration in the culture medium . Azurechelin enhanced the growth of P . cepacia in a medium containing transferrin 200 mg/L . Azurechelin released iron from transferrin in an equilibrium dialysis assay, suggesting that it could complete with transferrin for iron . Azurechelin could also stimulate iron uptake by P . cepacia . This siderophore appeared to have a novel structure with neither the typical characteristics of catechol nor of hydroxamate compounds.

J Bacteriol, 1992 Mar, 174(6), 1742 - 9
Phenotypic expression of the Pseudomonas syringae pv . syringae 61 hrp/hrm gene cluster in Escherichia coli MC4100 requires a functional porin; Li TH et al.; Plants, in general, appear to be able to detect the presence of incompatible Pseudomonas syringae strains by a hypothetical cell-cell recognition process to initiate inducible defense mechanisms that contribute to disease resistance . A 25-kb hrp/hrm gene cluster isolated from P . syringae pv . syringae 61(pHIR11) enables Escherichia coli to elicit a hypersensitive response (HR), a plant response generally considered to be a manifestation of recognition and resistance . To identify the nature of the HR-eliciting signal produced by E . coli cells carrying pHIR11, bacterial surface features were surveyed by immunological and biochemical procedures . No immunoreactive epitopes or outer membrane proteins were detected that were associated with expression of the P . syringae pv . syringae 61 hrp/hrm cluster in E . coli MC4100 . Phenotypic expression of the P . syringae pv . syringae 61 hrp/hrm cluster in E . coli MC4100, however, was found to be dependent upon ompC and ompF, which control outer membrane permeability to hydrophilic solutes . The results suggest that deployment of the HR-eliciting signal occurs via outer membrane porins and imply that a low-molecular-weight, hydrophilic factor mediates signal exchange between the bacterium and the responding plant cell.

Biochemistry, 1992 Feb 25, 31(7), 1961 - 8
1H NMR sequential assignments and identification of secondary structural elements in oxidized putidaredoxin, an electron-transfer protein from Pseudomonas; Ye XM et al.; Sequential 1H resonance assignments and secondary structural features of putidaredoxin (Pdx), a 106-residue globular protein consisting of a single polypeptide chain and a {2Fe-2S} cluster, are reported . No crystal structure has been obtained for Pdx or for any closely homologous protein . The sequentially assigned resonances represent ca . 83% of all the protons in Pdx and a large majority of those protons which are unaffected by the paramagnetism of the iron-sulfur cluster . A total of three alpha-helices, two beta-sheets, and two type I beta-turns have been identified from NOE (nuclear Overhauser effect) patterns . Besides the extensive beta-sheet described previously, a second sheet is identified, consisting of two short antiparallel strands (Ile 89-Thr 91 and Val 21-Leu 23), one of which ends in a tight type I turn (Thr 91-Pro 92-Glu 93-Leu 94) . One short helix (Ala 26-Gly 31) and a second longer helical region (Glu 54-Cys 73) are present . This second helical region is discontinuous, breaking at Pro 61, resuming at Glu 65, and ending at Cys 73 . The functionally important C-terminal tryptophan residue has been identified, and some structural constraints on this residue are described . Previously reported functional data concerning Pdx are discussed in light of present structural information . Finally, approaches to the determination of a high-resolution solution structure of the protein are discussed.

Carbohydr Res, 1992 Feb 7, 224, 1 - 17
Roles of potassium ions, acetyl and L-glyceryl groups in native gellan double helix: an X-ray study; Chandrasekaran R et al.; Native gellan, the natural form of the polysaccharide excreted by the bacterium Pseudomonas elodea, has a tetrasaccharide repeating unit that contains L-glycerol and acetate ester groups, and forms only weak and elastic gels . Based on X-ray diffraction data from well oriented and polycrystalline fibers of its potassium salt, the crystal structure of native gellan, including ions and water, has been determined and refined to a final R-value of 0.17 . The molecule forms of a half-staggered, parallel, double helix of pitch 5.68 nm which is stabilized by hydrogen bonds involving the hydroxymethyl groups in one chain and both carboxylate and glyceryl groups in other . Two molecules are packed in an antiparallel fashion in a trigonal unit cell of side a = 1.65 nm . Although the gross molecular morphology and packing arrangements are isomorphous with those observed in the crystal structure of potassium gellan, which is devoid of any substitutions, native gellan exhibits exceptional changes in its ion binding characteristics with respect to gellan . In particular, the L-glyceryl groups do not allow the gellan-like coordinated interactions of the ions and the carbohydrate groups, within and between double helices, which are necessary for strong gelation . These results at the molecular level explain, for the first time, the differences in the behavior of the polymer with and without substitutions.

J Thorac Cardiovasc Surg, 1992 Feb, 103(2), 295 - 306
Pulmonary transplantation . Early and late results . The Toronto Lung Transplant Group; de Hoyos AL et al.; Between November 1983 and March 1991, we performed 50 single and 40 double lung transplants in 82 recipients . Early deaths occurred in six (13%) single and in eight (21%) double lung transplant recipients . Late deaths occurred in 11 (28%) single and in one (3%) double lung recipients . Twenty-three of 37 (62%) single and 17 of 24 (71%) double lung transplant recipients have survived at least 1 year after the operation . In patients surviving at least 3 months after the operation (36 of 47 single lung transplant {77%} and 28 of 37 double lung transplant recipients {76%}), significant improvement occurred in arterial blood gases, pulmonary function tests, and exercise capacity . During our initial experience, airway anastomotic complications were the main cause of early morbidity and mortality . With newer surgical techniques and improved perioperative care, airway complications are now uncommon . Infectious complications, either bacterial (Pseudomonas cepacia) or viral (cytomegalovirus), are now the main cause of early mortality . Chronic rejection in the form of obliterative bronchiolitis has become a frequent cause of late morbidity.

J Thorac Cardiovasc Surg, 1992 Feb, 103(2), 287 - 93; discussion 294
Bilateral lung transplantation for cystic fibrosis . The Toronto Lung Transplant Group; Ramirez JC et al.; Between March 1988 and March 1991, 17 patients underwent bilateral lung transplantation for end-stage lung disease caused by cystic fibrosis . There were 11 male and six female patients . Ages ranged from 19 to 41 years (mean age 28 years) . Preoperative mean arterial oxygen tension with the patient breathing room air was 54 +/- 6 mm Hg; forced vital capacity, 1.8 +/- 0.7 L; forced expiratory volume in 1 second, 0.9 +/- 0.3 L; and 6-minute walk test, 506 +/- 44 m . Immunosuppression consisted of cyclosporine, azathioprine, and prednisone . Induction immunosuppression was obtained with Minnesota antilymphocyte globulin . All patients received perioperative antibiotics according to sputum cultures and sensitivities . There were six operative deaths, four of which resulted from bacterial infection . Two patients required a second transplantation, one receiving a single lung and one undergoing bilateral lung replacement . Significant functional improvement was observed in all survivors . At 3 months follow-up, mean arterial oxygen tension on room air was 95 +/- 6 mm Hg (p less than 0.01); forced vital capacity, 3 +/- 0.8 L (p less than 0.01); forced expiratory volume in 1 second, 2.6 +/- 0.9 L (p less than 0.01); and 6-minute walk test, 678 +/- 47 m (p less than 0.01) . The actuarial survival rate was 66% at 3 months and 58% at 6, 12, and 24 months . The most frequent cause of morbidity and mortality was acute pneumonia resulting from Pseudomonas cepacia . For patients with respiratory failure caused by cystic fibrosis, bilateral lung transplantation is an effective treatment option associated with significant functional improvement.

Chest, 1992 Feb, 101(2), 555 - 7
Ceftazidime monotherapy for pulmonary melioidosis in a traveler returning from Thailand; Sauerwein RW et al.; A patient with deteriorating pulmonary melioidosis rapidly recovered after treatment with ceftazidime . To prevent possible relapses, an oral maintenance regimen of amoxicillin and clavulanic acid was prescribed for a period of three months . Melioidosis is caused by Pseudomonas pseudomallei . It is an insidious disease because of its variable clinical presentation, possible long-term asymptomatic carriage, broad-spectrum resistance to first-line antibiotics, and high mortality rate . As in our patient, the diagnosis should be particularly considered when there is reduced immunologic resistance and previous exposure in endemic areas, such as Southeast Asia.

J Bacteriol, 1992 Feb, 174(3), 962 - 9
Cloning and sequencing of the genes involved in the conversion of 5-substituted hydantoins to the corresponding L-amino acids from the native plasmid of Pseudomonas sp . strain NS671; Watabe K et al.; Pseudomonas sp . strain NS671, which produces L-amino acids asymmetrically from the corresponding racemic 5-substituted hydantoins, harbored a plasmid of 172 kb . Curing experiments suggest that this plasmid, designated pHN671, is responsible for the conversion of 5-substituted hydantoins to their corresponding L-amino acids by strain NS671 . DNA fragments containing the genes involved in this conversion were cloned from pHN671 in Escherichia coli by using pUC18 as a cloning vector . The smallest recombinant plasmid, designated pHPB12, contained a 7.5-kb insert DNA . The nucleotide sequence of the insert DNA was determined, and three closely spaced open reading frames predicted to encode peptides with molecular masses of 75.6, 64.9, and 45.7 kDa were found . These open reading frames were designated hyuA, hyuB, and hyuC, respectively . Cell extracts from E . coli carrying deletion derivatives of pHPB12 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gene products of hyuA, hyuB, and hyuC were identified . The functions of these gene products were also examined with the deletion derivatives . The results indicate that both hyuA and hyuB are involved in the conversions of D- and L-5-substituted hydantoins to corresponding N-carbamyl-D- and N-carbamyl-L-amino acids, respectively, and that hyuC is involved in the conversion of N-carbamyl-L-amino acids to L-amino acids.

Infect Immun, 1992 Feb, 60(2), 497 - 502
Characterization of a cellular protease that cleaves Pseudomonas exotoxin; Fryling C et al.; Pseudomonas exotoxin (PE) is a 66-kDa bacterial toxin that is proteolytically cleaved by cells to produce an N-terminal fragment of 28 kDa and a C-terminal 37-kDa fragment which translocates to the cytosol and inhibits protein synthesis (M . Ogata, V.K . Chaudhary, I . Pastan, and D.J . FitzGerald, J . Biol . Chem . 265:20678-20685, 1990) . When cells were broken by homogenization, the appropriate proteolytic activity was found associated with cellular membranes and not in a soluble fraction . Proteolysis of PE by crude membranes was stimulated by divalent cations, was ATP independent, and had a pH optimum of 5.5 . When cells were disrupted by nitrogen cavitation and fractionated on Percoll gradients, proteolytic activity was present in fractions corresponding to the density of plasma membranes or endosomes but not in fractions containing lysosomes . Proteolytic activity was recovered in detergent extracts after crude membranes were treated with Nonidet P-40 or octylglucoside . Proteolysis of PE by either crude membranes or detergent extracts generated fragments of 28 and 37 kDa . The sizes of these fragments resembled those produced by intact cells . However, when the nontoxic mutant, PEgly276, which cannot be cleaved appropriately by intact cells, was incubated with membranes or extracts there was no production of the 28- and 37-kDa fragments.

Appl Environ Microbiol, 1992 Feb, 58(2), 744 - 6
Effect of nitrogen limitation on long-side-chain poly-beta-hydroxyalkanoate synthesis by Pseudomonas resinovorans; Ramsay BA et al.; Pseudomonas resinovorans produced poly-beta-hydroxyalkanoates (PHAs) when grown on hydrocarbons but not on glucose . In a chemostat culture, the PHA composition was beta-hydroxybutyrate (C4)-beta-hydroxyhexanoate (C6)-beta-hydroxyoctanoate (C8)-beta-hydroxydecanoate (C10) (1:15:75:9) on octanoate and C4-C6-C8-C10 (8:62:23:7) on hexanoic acid . Contrary to the reported behavior of Pseudomonas oleovorans, the PHA accumulation rate increased under ammonium limitation on octanoate.

Appl Environ Microbiol, 1992 Feb, 58(2), 450 - 4
Enzymatic iron oxidation by Leptothrix discophora: identification of an iron-oxidizing protein; Corstjens PL et al.; An iron-oxidizing factor was identified in the spent culture medium of the iron- and manganese-oxidizing bacterial strain Leptothrix discophora SS-1 . It appeared to be a protein, with an apparent molecular weight of approximately 150,000 . Its activity could be demonstrated after fractionation of the spent medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . A spontaneous mutant of L . discophora SS-1 was isolated which excreted neither manganese- nor iron-oxidizing activity, whereas excretion of other proteins seemed to be unaffected . Although the excretion of both metal-oxidizing factors was probably linked, the difference in other properties suggests that manganese and iron oxidation represent two different pathways . With a dot-blot assay, it was established that different bacterial species have different metal-oxidizing capacities . Whereas L . discophora oxidized both iron and manganese, Sphaerotilus natans oxidized only iron and two Pseudomonas spp . oxidized only manganese.

Pediatr Emerg Care, 1992 Feb, 8(1), 38 - 44
An update on the evaluation and management of plantar puncture wounds and Pseudomonas osteomyelitis; Inaba AS et al.; The management of children who present to the ED with plantar puncture wounds is dependent upon the nature of the injury, the examination of the puncture site, and the potential risk of a retained foreign body . Not all patients will require wound enlargement and a search for a retained foreign body . Close follow-up of all children who are being treated as outpatients is of vital importance in detecting an early development of an infectious complication . Pseudomonas osteomyelitis should be suspected in all patients who present with foot pain, swelling, and a decreased ability to bear weight after sustaining a nail puncture through a sneaker . The current consensus favors open surgical debridement followed by a course of intravenous antibiotics . The exact duration of the postoperative antibiotic course is still being debated.

Clin Nucl Med, 1992 Feb, 17(2), 90 - 3
Aerosol scintigraphy in the assessment of therapy for cystic fibrosis; Kuni CC et al.; Thirteen patients with cystic fibrosis (aged 11 to 32 years) who were hospitalized for exacerbation and who had sputum cultures positive for Pseudomonas organisms were treated initially for 4 days with bronchodilators and physiotherapy followed by the addition of antibiotic (14 days, n = 8) or placebo (14 days, n = 4; 7 days, n = 1) . Tc-99m DTPA aerosol scintigraphy was performed on the day before bronchodilators and physiotherapy, on the day before antibiotic or placebo, and on the day after completion of antibiotic or placebo therapy . Scintigrams were evaluated for change in the number of nonventilated segments and change in the number of bronchial deposits of aerosol . Sixty-nine percent of patients showed improvement after bronchodilators and physiotherapy alone . Sixty-two percent showed further improvement after antibiotic or placebo was added; this improvement was independent of whether antibiotic or placebo was administered (P greater than 0.1) . These aerosol scintigraphy results failed to demonstrate that the effectiveness of bronchodilators and physiotherapy is enhanced by antibiotics in the treatment of cystic fibrosis exacerbations.

Clin Infect Dis, 1992 Feb, 14(2), 412 - 7
Pseudomonas pseudomallei liver abscesses: a clinical, laboratory, and ultrasonographic study; Vatcharapreechasakul T et al.; Ultrasonography revealed evidence of liver abscess in 126 patients who were admitted to one hospital in northeastern Thailand over a 3-year period . There were 50 cases for which a pyogenic bacterial etiology was confirmed; 34 cases (group 1) were caused by Pseudomonas pseudomallei (nine patients died) and 16 cases (group 2) were caused by other bacteria (two patients died) . Melioidosis was associated with anemia and underlying diabetes or renal disease; right-upper-quadrant pain and jaundice were more common in group 2 (P less than .05) . Blood cultures were positive for bacteria in 68% of group 1 and 50% of group 2 . Chest radiographs revealed abnormalities in 17 of 30 group 1 patients and 6 of 12 group 2 patients . The radiographic appearances of a blood-borne pneumonia suggested melioidosis . The serum indirect hemagglutination assay for antibodies to P . pseudomallei was of limited value in differentiating the two types of abscesses . Multiple hypoechoic areas on ultrasonography were significantly associated with melioidosis (P less than .01); associated splenic abscess occurred in 19 group 1 patients but only one group 2 patient (2-107, 95% confidence interval; odds ratio, 19) . In an area where P . pseudomallei is endemic, these characteristic ultrasonographic findings should prompt immediate treatment for melioidosis.

Ophthalmic Surg, 1992 Feb, 23(2), 137 - 9
Penetrating keratoplasty for corneal perforation in an obtunded patient; Van Meter WS et al.; A ventilator-dependent patient obtunded from severe head trauma suffered a spontaneous corneal perforation with lens extrusion secondary to nosocomial Pseudomonas keratitis . Despite the patient's guarded condition, a successful tectonic penetrating keratoplasty with lens removal was performed for restoration of the globe . Upon recovery, the patient's only useful vision was in her operated eye . Preventative measures against prolonged corneal exposure in an obtunded patient include copious artificial tears and lubricants, use of scleral lenses, moisture chambers, bandage contact lenses, or tarsorrhaphies.

Can J Microbiol, 1992 Feb, 38(2), 111 - 4
Epiphytic populations of Pseudomonas syringae on barley; Georgakopoulos DG et al.; The epiphytic populations of Pseudomonas syringae were monitored on 23 barley entries planted in the field in four replications during the summer of 1986, and on six selected entries during the summer of 1987, from the second-leaf stage until senescence . Populations were initially low (0-3 log colony-forming units (cfu) per leaf) in all but one entry; they generally increased throughout the season, and at the end they reached 3-7 log cfu/leaf . Significant differences among the average epiphytic populations were found in the 1986 trial; only one entry, however, had a significantly different average population in the 1987 trial . The slopes of population increase were also compared: significant differences were observed in 1986 but not in 1987 . In addition to epiphytic population counts, the percentage of ice nucleation active bacteria was determined in the population isolated from each leaf sample, and averaged throughout the season for each entry . Significant differences were observed in 1986 and in 1987 . When the entries were ranked according to their average epiphytic population and compared between the two experiments, they were found to be very similar . The same was not true for the other parameters studied in the experiment.

Zhonghua Yi Xue Za Zhi, 1992 Feb, 72(2), 74 - 6, 126-7
{Characteristics of monoclonal antibodies against chorionic gonadotropin receptor}; Han S; We reported the production of monoclonal antibodies (McAb) against chorionic gonadotropin (CG) receptor by fusing spleen cells of BALB/c mice immunized with purified pseudomonas maltophilia CG receptor with mouse myeloma line (SP 2/0) . Four hybridoma cell lines secreting CG receptor McAbs were obtained (ED 490, DG 390, AB 890, GE 590) . GE 590 is IgG 1; ED 490, DG 390, AB 890 are IgG2b . I125-HCG and ED 490, DG 390 . AB 890 recognized CG receptor different antigenic determinant . Increased concentrations of GE 590 were in inverse proportion to the amount of I125-HCG binding to human ovarian tissues showing that normal ovarian tissues and ovarian malignant tumors have different HCG receptor antigenic determinant . This study indicated that these McAbs may be useful in studying the structure of CG receptor and in providing a valuable evidence for early diagnosis and treatment of ovarian malignant tumors.

Jpn J Med Sci Biol, 1992 Feb, 45(1), 35 - 47
Endotoxin of Pseudomonas pseudomallei detected by the body weight-decreasing reaction in mice and comparison of it with those of P . cepacia and P . aeruginosa; Iwasa S et al.; The heat-treated whole cells, culture supernatants, and extracted endotoxin preparations of Pseudomonas pseudomallei were examined for endotoxin by the mouse body weight-decreasing (BWD) test . The experiments were conducted also with those of P . cepacia and P aeruginosa . Endotoxin was detected in all the samples of P . pseudomallei . Endotoxin of P . cepacia was detected in whole cells, but not in culture supernatant . The BWD activity of P . aeruginosa was 30 times as high as that of P . pseudomallei . This result was confirmed by the experiments with endotoxin preparations . In the limulus amebocyte lysate gelation (LAL) test, however, the endotoxin preparations of the two species showed the same level of activity.

Am J Trop Med Hyg, 1992 Feb, 46(2), 151 - 7
Serial serum C-reactive protein levels as an aid to the management of melioidosis; Ashdown LR; Of 46 patients with clinical melioidosis, 35 (22 culture-positive and 13 culture-negative) had relatively uneventful disease courses, with elevated serum C-reactive protein (CRP) concentrations (greater than 5 mg/dl) that decreased with the commencement of appropriate antibiotic therapy, and continued to show an uninterrupted decrease (mean 29.4 days, range 12-52 days) to the normal range (less than 1 mg/dl), with resolution of their infections . In five culture-positive patients with complicated disease courses, CRP concentrations remained elevated (greater than 5 mg/dl) until the underlying disorders were successfully managed, or until the antibiotic regimen was changed, and CRP values then decreased to the normal range . During surveillance, elevated CRP concentrations (greater than 10 mg/dl) led to the diagnosis of reactivation of infection in three afebrile patients, while the serum CRP values in other patients remained within the normal range in the absence of intercurrent complications . The CRP estimations may be helpful in ascertaining active infection in patients with low serum levels of specific IgM antibody, and serial measurements of serum CRP in patients with clinical melioidosis may be useful in determining the optimal duration of treatment and for detecting occult or unresolved infection with Pseudomonas pseudomallei.

Protein Sci, 1992 Feb, 1(2), 259 - 70
Substrate polarization by residues in delta 5-3-ketosteroid isomerase probed by site-directed mutagenesis and UV resonance Raman spectroscopy; Austin JC et al.; delta 5-3-Ketosteroid isomerase (KSI: EC 5.3.3.1) of Pseudomonas testosteroni catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by the stereospecific transfer of the steroid 4 beta-proton to the 6 beta-position, using Tyr-14 as a general acid and Asp-38 as a base . Ultraviolet resonance Raman (UVRR) spectra have been obtained for the catalytically active double mutant Y55F + Y88F, which retains Tyr-14 as the only tyrosine residue (referred to as the Y14(0) mutant), and the Y14F mutant, which has 50,000-fold lower activity . The UVRR results establish that binding of the product analog and competitive inhibitors 19-nortestosterone or 4-fluoro-19-nortestosterone to the Y14(0) mutant does not result in the formation of deprotonated Tyr-14 . The UVRR spectra of the steroid inhibitors show large decreases in the vinyl and carbonyl stretching frequencies on binding to the Y14(0) enzyme but not on binding to the Y14F enzyme . These changes cannot be mimicked by protonation of the steroids . For 19-nortestosterone, the vinyl and carbonyl stretching frequencies shift down (with respect to the values in aqueous solution) by 18 and 27 cm-1, respectively, on binding to Y14(0) KSI . It is proposed that the changes in the steroid resonance Raman spectrum arise from polarization of the enone moiety via the close proximity of the charged Asp-38 side chain to the vinyl group and the directional hydrogen bond between Tyr-14 and the 3-carbonyl oxygen of the steroid enone . The 230-nm-excited UVRR spectra do not, however, show changes that are characteristic of strong hydrogen bonding from the tyrosine hydrogen . It is proposed that this hydrogen bonding is compensated by a second hydrogen bond to the Tyr-14 oxygen from another protein residue . UVRR spectra of the Y14(0) enzyme obtained using 200 nm excitation show enhancement of the amide II and S Raman bands . The secondary structure of KSI was estimated from the amide II and S intensities and was found to be low in alpha-helical structure . The alpha-helix content was estimated to be in the range of 0-25% (i.e., 10 +/- 15%).

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