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