Pseudomonas

Adv Perit Dial, 1991, 7, 102 - 4
Successful treatment of tuberculous peritonitis while maintaining patient on CAPD; Tan D et al.; Although conventional wisdom advises removal of the Tenckhoff catheter as part of the therapy for tuberculous peritonitis, there are a few recent reports of cases successfully treated while maintaining the patients on CAPD . We wish to report three cases treated without interrupting CAPD . In two of the patients, cultures were positive for Mycobacterium tuberculosis and in the third case, although the cultures were negative, the patient improved on anti-Tb medications . Smear for AFB was positive in one patient; and two had a positive PPD . All had predominance of lymphocytes and monocytes in effluent . The total WBC count was 160-300 and two patients had fever . All had abdominal pain . One patient was treated with INH and ethambutol; one with INH and rifampin and one (who was suspected of being HIV+) also received pyrazinamide (PZA) until culture was available . Cultures grew in 4-6 weeks . All were started on therapy prior to having the culture results, and all showed clinical improvement within two weeks . One patient had his catheter replaced two months later because of pseudomonas peritonitis, continued on CAPD for an additional five months, then changed to HD because of recurrent bacterial peritonitis . One patient died of complications of diabetic vascular disease three months later with no evidence of peritonitis . One patient has remained on anti-Tb treatment for seven months and is doing well on CAPD.

Life Sci, 1991, 49(5), 367 - 74
Prophylaxis against organophosphate poisoning by an enzyme hydrolysing organophosphorus compounds in mice; Ashani Y et al.; Parathion hydrolase purified from Pseudomonas sp . was injected i.v . into mice to demonstrate the feasibility of using organophosphorus acid anhydride (OPA) hydrolases as pretreatment against organophosphates (OP) poisoning . Results show that exogenous administration of as low as 7 to 26 micrograms of parathion hydrolase conferred protection against challenge with multiple median lethal doses (LD50) of diethyl p-nitrophenyl phosphate (paraoxon; 3.8-7.3 x LD50) and diethylfluorophosphate (DEFP; 2.9 x LD50) without administration of supportive drugs . The extent of protection observed was consistent with blood-parathion hydrolase levels and the kinetic constants of the enzymatic hydrolysis of paraoxon and DEFP by parathion hydrolase . OPA hydrolases not only appear to be potential prophylactic drugs capable of increasing survival ratio following OP intoxication but also to alleviate post-exposure symptoms.

Biodegradation, 1991-92, 2(3), 165 - 70
Toxicity of chlorobenzene on Pseudomonas sp . strain RHO1, a chlorobenzene-degrading strain; Fritz H et al.; Pseudomonas sp . strain RHO1 able to use chloro- and 1,4-dichlorobenzene as growth substrates was tested towards sensitivity against chlorobenzene . Concentrations of chlorobenzene higher than 3.5 mM were found to be toxic to cells independent of pregrowth with chlorobenzene or nutrient broth . Below this concentration, sensitivity towards chlorobenzene depended on the precultivation of the cells, i.e . type of growth substrate (chlorobenzene or nutrient broth) and the concentration of chlorobenzene as the growth substrate . Cells grown in continuous culture were especially sensitive with a threshold concentration of 2.5 mM chlorobenzene . In addition to chlorobenzene, metabolites also seem to function as toxic compounds . 2-Chlorophenol and 3-chlorocatechol were isolated from cell extracts . Cleavage of 3-chlorocatechol by catechol 1,2-dioxygenase seems to be the critical step in the metabolism of chlorobenzene.

Biodegradation, 1991, 2(2), 115 - 20
Metabolism of naphthalene by the biphenyl-degrading bacterium Pseudomonas paucimobilis Q1; Kuhm AE et al.; Pseudomonas paucimobilis Q1 originally isolated as biphenyl degrading organism (Furukawa et al . 1983), was shown to grow with naphthalene . After growth with biphenyl or naphthalene the strain synthesized the same enzyme for the ring cleavage of 2,3-dihydroxybiphenyl or 1,2-dihydroxynaphthalene . The enzyme, although characterized as 2,3-dihydroxybiphenyl dioxygenase (Taira et al . 1988), exhibited considerably higher relative activity with 1,2-dihydroxynaphthalene . These results demonstrate that this enzyme can function both in the naphthalene and biphenyl degradative pathway.

Appl Microbiol Biotechnol, 1991 Jan, 34(4), 556 - 7
Degradation of isomeric monochlorobenzoates and 2,4-dichlorophenoxyacetic acid by a constructed Pseudomonas sp; Sahasrabudhe AV et al.; A 4-chlorobenzoate-degrading Pseudomonas sp . US1 was mated with a strain of Escherichia coli JMP 397 (harbouring the plasmid pJP4) . An ex-conjugant designated Pseudomonas sp . US1 ex that could utilize all the isomeric monochlorobenzoates and 2,4-dichlorophenoxyacetate was obtained . The ex-conjugant released stoichiometric amounts of chloride when grown on these chloroaromatics as sole sources of carbon and energy.

Biodegradation, 1991-92, 2(4), 245 - 52
Growth and survival of Pseudomonas cepacia DBO1 (pRO101) in soil amended with 2,4-dichlorophenoxyacetic acid; Jacobsen CS et al.; The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading pseudomonad, Pseudomonas cepacia DBO1(pRO101), was inoculated at approximately 10(7) CFU/g into sterile and non-sterile soil amended with 0, 5 or 500 ppm 2,4-D and the survival of the strain was studied for a period of 44 days . In general, the strain survived best in sterile soil . When the sterile soil was amended with 2,4-D, the strain survived at a significantly higher level than in non-amended sterile soil . In non-sterile soil either non-amended or amended with 5 ppm 2,4-D the strain died out, whereas with 500 ppm 2,4-D the strain only declined one order of magnitude through the 44 days . The influence of 0, 0.06, 12 and 600 ppm 2,4-D on short-term (48 h) survival of P . cepacia DBO1(pRO101) inoculated to a level of 6 x 10(4), 6 x 10(6) or 1 x 10(8) CFU/g soil was studied in non-sterile soil . Both inoculum level and 2,4-D concentration were found to have a positive influence on numbers of P . cepacia DBO1(pRO101) . At 600 ppm 2,4-D growth was significant irrespective of the inoculation level, and at 12 ppm growth was stimulated at the two lowest inocula levels . P . cepacia DBO1(pRO101) was able to survive for 15 months in sterile buffers kept at room temperature . During this starvation, cells shrunk to about one third the volume of exponentially growing cells.

J Biol Chem, 1990 Dec 25, 265(36), 22187 - 96
Gentisate 1,2-dioxygenase from Pseudomonas . Substrate coordination to active site Fe2+ and mechanism of turnover; Harpel MR et al.; Gentisate 1,2-dioxygenase catalyzes the oxygenolytic ring cleavage of gentisate (2,5-dihydroxybenzoate) between carbons 1 and 2 to form maleylpyruvate . The essential active site Fe2+ of the enzyme binds NO to yield an EPR-active (S = 3/2) complex . Hyperfine broadening from 17O (I = 5/2) is observed in the spectrum of the enzyme-nitrosyl complex prepared in 17O-enriched water, demonstrating that water is an iron ligand . Association of gentisate with the enzyme-nitrosyl complex causes the broadening due to {17O}water to disappear, suggesting that water is displaced . Hyperfine broadening of the EPR spectrum for the gentisate-bound complex is observed when 17O is incorporated into either the carbon 1 carboxylate or carbon 2 hydroxyl substituents of gentisate, but not when it is placed in the carbon 5 hydroxyl substituent . Thus, substrate apparently binds directly to the iron through the carbon 1 carboxylate and carbon 2 hydroxyl substituents, thereby bringing the site of ring cleavage close to the active site iron . Since NO must bind to the iron to elicit an EPR signal, a total of three sites in the iron coordination appear to be available for exogenous ligands . The role of the substrate functional groups in catalysis is investigated through comparison of the reaction kinetics of gentisate analogs using the gentisate 1,2-dioxygenases isolated from Pseudomonas acidovorans and Pseudomonas testosteroni . Turnover is either eliminated or substantially reduced on replacement of any of the functional groups of gentisate . Furthermore, an electron-donating group that can tautomerize (hydroxyl or amine) is required in a ring position either ortho or para to the carbon 2 substituent for turnover . The best alternate substrate of this group is 5-aminosalicylate, which is turned over at approximately 7% of the rate of gentisate by the enzyme from P . testosteroni . Both atoms from O2 are shown to be incorporated into the product of 5-aminosalicylate turnover . This is the first direct demonstration of dioxygenase stoichiometry in the reaction of any ferrous, non-heme, aromatic ring-cleaving dioxygenase . It is proposed that the enzyme-catalyzed O2 attack on the aromatic ring of gentisate is initiated from a complex in which O2 and substrate are simultaneously coordinated to the active site iron . Subsequent dioxygen bond cleavage and insertion are proposed to be promoted by a resonance shift involving ketonization of the carbon 5 hydroxyl group.

Cancer Res, 1990 Dec 15, 50(24), 7786 - 8
Expression of the interleukin 6 receptor and interleukin 6 in prostate carcinoma cells; Siegall CB et al.; We have probed for the presence of interleukin 6 (IL6) receptors in prostatic carcinoma cell lines (LNCaP, DU 145, and PC3) by examining their sensitivity to the cytotoxic effects of a chimeric toxin composed of IL6 and Pseudomonas exotoxin (PE) . All three cell lines were killed by IL6-PE66(4)Glu, a version of IL6-PE in which the binding domain of native PE has been mutated to debilitate PE binding to its own receptor . This cytotoxic activity confirmed the presence of IL6 receptors on prostatic carcinoma cells . We have measured the number of IL6 receptors found on these cells and have further determined that they secrete IL6 . These data provide evidence that IL6 and its receptor may play an important role in human prostate cancer.

J Biol Chem, 1990 Dec 15, 265(35), 21498 - 503
Chemical and kinetic evidence for an essential histidine in the phosphotriesterase from Pseudomonas diminuta; Dumas DP et al.; The pH rate profile for the hydrolysis of diethyl-p-nitrophenyl phosphate catalyzed by the phosphotriesterase from Pseudomonas diminuta shows a requirement for the deprotonation of an ionizable group for full catalytic activity . This functional group has an apparent pKa of 6.1 +/- 0.1 at 25 degrees C, delta Hion of 7.9 kcal/mol, and delta Sion of -1.4 cal/K.mol . The enzyme is not inactivated in the presence of the chemical modification reagents dithiobis-(2-nitrobenzoate), methyl methane thiosulfonate, carbodiimide, pyridoxal, butanedione, or iodoacetic acid and thus cysteine, asparate, glutamate, lysine, and arginine do not appear to be critical for catalytic activity . However, the phosphotriesterase is inactivated completely with methylene blue, Rose Bengal, or diethyl pyrocarbonate . The enzyme is not inactivated by diethyl pyrocarbonate in the presence of bound substrate analogs, and inactivation with diethyl pyrocarbonate is reversible upon addition of neutralized hydroxylamine . The modification of a single histidine residue by diethyl pyrocarbonate, as shown by spectrophotometric analysis, is responsible for the loss of catalytic activity . The pKinact for diethyl pyrocarbonate modification is 6.1 +/- 0.1 at 25 degrees C . These results have been interpreted to suggest that a histidine residue at the active site of phosphotriesterase is facilitating the reaction by general base catalysis.

J Biol Chem, 1990 Dec 15, 265(35), 21634 - 41
Identification of the principal catalytically important acidic residue of 3-hydroxy-3-methylglutaryl coenzyme A reductase; Wang Y et al.; Kinetic analysis of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase has implicated a glutamate or aspartate residue in (i) formation of mevaldate thiohemiacetal by proton transfer to the carbonyl oxygen of mevaldate and (ii) enhanced ionization of CoASH by the resulting enzyme carboxylate anion, facilitating attack by CoAS- on the carbonyl carbon of mevaldate (Veloso, D., Cleland, W . W., and Porter, J . W . (1981) Biochemistry 81, 887-894) . Although neither the identity of this acidic residue nor its location is known, the catalytic domains of 11 sequenced HMG-CoA reductases contain only 3 conserved acidic residues . For HMG-CoA reductase of Pseudomonas mevalonii, these residues are Glu52, Glu83, and Asp183 . To identify the acidic residue that functions in catalysis, we generated mutants having alterations in these residues . The mutant proteins were expressed, purified, and characterized . Mutational alteration of residues Glu52 or Asp183 of P . mevalonii HMG-CoA reductase yielded enzymes with significant, but in some cases reduced, activity (Vmax = 100% Asp183----Ala, 65% Asp183----Asn, and 15% Glu52----Gln of wild-type activity, respectively) . Although the activity of mutant enzymes Glu52----Gln and Asp183----Ala was undetectable under standard assay conditions, their Km values for substrates were 4-300-fold higher than those for wild-type enzyme . Km values for wild-type enzyme and for mutant enzymes Glu52----Gln and Asp183----Ala were, respectively: 0.41, 73, and 120 mM {R,S)-mevalonate); 0.080, 4.4, and 2.0 mM (coenzyme A); and 0.26, 4.4, and 1.0 mM (NAD+) . By these criteria, neither Glu52 nor Asp183 is the acidic catalytic residue although each may function in substrate recognition . During chromatography on coenzyme A agarose or HMG-CoA agarose, mutant enzymes Asp183----Asn and Glu83----Gln behaved like wild-type enzyme . By contrast, and in support of a role for these residues in substrate recognition, mutant enzymes Glu52----Gln and Asp183----Ala exhibited impaired ability to bind to either support . Despite displaying Km values for substrates and chromatographic behavior on substrate affinity supports comparable to wild-type enzyme, only mutant enzyme Glu83----Gln was essentially inactive under all conditions studied (Vmax = 0.2% that of wild-type enzyme) . Glutamate residue 83 of P . mevalonii HMG-CoA reductase, and consequently the glutamate of the consensus Pro-Met-Ala-Thr-Thr-Glu-Gly-Cys-Leu-Val-Ala motif of the catalytic domains of eukaryotic HMG-CoA reductases, is judged to be the acidic residue functional in catalysis.

J Antimicrob Chemother, 1990 Dec, 26 Suppl F, 25 - 9
Tolerance and safety of ciprofloxacin in paediatric patients; Black A et al.; The Cystic Fibrosis Clinic at the Royal Belfast Hospital for Sick Children has treated 31 children with ciprofloxacin, for serious pseudomonas infection in cystic fibrosis, and carefully monitored the safety and acceptability of the drug . Initially, eight very ill children were treated on a named-patient basis, with an encouraging clinical response and few adverse effects . Children aged 10-18 years were included in a study of four consecutive exacerbations of respiratory disease, comparing (i) oral ciprofloxacin in each episode with (ii) ciprofloxacin alternating with intravenous azlocillin and tobramycin . Other children with cystic fibrosis were subsequently treated with ciprofloxacin, as the need arose . In all the groups very few adverse reactions were found; in particular only one child developed arthralgia . A total of 202 children in the UK have been treated with ciprofloxacin on a named-patient basis, and their clinicians have reported 46 adverse events that may have been drug-related . Overall ciprofloxacin appears to be safe and effective in children but concern about the possible occurrence of arthropathy remains and long term follow-up of these children may be necessary.

Arch Biochem Biophys, 1990 Dec, 283(2), 542 - 5
Isolation and characterization of a second molybdopterin dinucleotide: molybdopterin cytosine dinucleotide; Johnson JL et al.; The pterin cofactor (bactopterin) in the molybdoenzyme CO dehydrogenase isolated from Pseudomonas carboxydoflava has previously been shown to differ from molybdopterin in molecular mass, phosphate content, stability, and other properties, implying a novel structure . The structure of the CO dehydrogenase pterin has been investigated in the present studies by alkylation and isolation of the carboxamidomethyl derivative . The alkylated pterin was identified as {di-(carboxamidomethyl)}molybdopterin cytosine dinucleotide on the basis of its absorption properties and by degradation with nucleotide pyrophosphatase yielding carboxamidomethylmolybdopterin and CMP . Further treatment of these products with alkaline phosphatase produced species with absorption and chromatographic properties identical to those of the corresponding dephospho compounds . Molybdopterin cytosine dinucleotide is the second molybdopterin variant to be structurally characterized . The fact that molybdopterin cytosine dinucleotide and molybdopterin guanine dinucleotide contain molybdopterin in their structure shows that the pterin moiety, with its unique dithiolene-containing sidechain, is a structural element which is common to the organic portion of the molybdenum cofactors of many molybdoenzymes.

Toxicol Lett, 1990 Dec, 54(2-3), 157 - 67
Subacute inhalation toxicity study of an ice-nucleation-active Pseudomonas syringae administered as a respirable aerosol to rats; Goodnow RA et al.; The inhalation toxicity of a commercial sample of an ice-nucleation-active Pseudomonas syringae (strain 31a) was evaluated by repetitively exposing rats to about 700 mg/m3 of an aerosol consisting of a suspension of 0.0008, 0.4 or 0.8 g/l of bacteria in water for 2 h per day, 5 days per week for 13-14 exposures . No mortality, moribundity or biologically significant differences in clinical signs, body weight, food consumption or clinical pathology were observed . Animals tested at 500 times (0.4 g/l) and 1000 times (0.8 g/l) the recommended ice-nucleation concentration (0.0008 g/l) exhibited concentration-dependent increased lung weights . Several animals exhibited enlarged tracheobronchial lymph nodes . The pulmonary responses observed are considered compatible with a mild irritant reaction . There was no evidence of bacterial infection . Animals tested at a concentration typical for the discharge mouth of a snow gun (0.0008 g/l) demonstrated no significant biological effect.

J Med Microbiol, 1990 Dec, 33(4), 265 - 9
3-deoxy-D-manno-2-octulosonic acid in the lipopolysaccharide of various strains of Pseudomonas cepacia; Straus DC et al.; Six clinical isolates of Pseudomonas cepacia (representing the five serotypes of the organism) were examined for the presence of 3-deoxy-D-manno-2-octulosonic acid (KDO) in their lipopolysaccharide (LPS) . Purified LPS was examined for the presence of KDO by the thiobarbituric acid (TBA) assay and by gas chromatography . All strains possessed KDO . One strain possessed KDO that was detectable by the TBA assay after mild acid hydrolysis with 0.04 M H2SO4 at 100 degrees C for 20 min . The other strains also possessed KDO but it was only demonstrable by the TBA assay after strong acid hydrolysis (4 M HCl for 60 min at 100 degrees C) . All six purified LPS preparations were shown to possess KDO by two separate gas chromatography procedures . LPS isolated from the six strains of P . cepacia was toxic for mice.

Br J Cancer, 1990 Dec, 62(6), 909 - 14
Disposition of the prodrug 4-(bis (2-chloroethyl) amino) benzoyl-L-glutamic acid and its active parent drug in mice; Antoniw P et al.; A novel therapy for improving selectivity in cancer chemotherapy aims to modify distribution of a cytotoxic drug by generating it selectively at tumour sites . In this approach an antibody-enzyme conjugate is allowed to localise at the tumour sites before injecting a prodrug which is converted to an active drug specifically by the targeted enzyme in the conjugate . We present here pharmacokinetic studies on the prodrug 4-(bis (2-chloroethyl) amino) benzoyl-L-glutamic acid and its activated derivative, benzoic acid mustard . The glutamic acid is cleaved from the prodrug to form the active drug by carboxypeptidase G2 (CPG2), an enzyme from Pseudomonas sp., which is not found in mammalian cells . The prodrug and its parent active drug were rapidly distributed in plasma and tissues after administration of prodrug or active drug (41 mumol kg-1 intraperitoneally) to mice bearing human choriocarcinoma xenografts . Prodrug and active drug both followed a two-compartment kinetic model . Prodrug was eliminated more rapidly (t1/2 alpha = 0.12 h, t1/2 beta = 0.70 h) than active drug (t1/2 alpha = 0.37 h, t1/2 beta = 1.61 h) . Conversion of the prodrug to the activated parent drug was detected within 5 min of administration to mice which had previously received a F(ab')2-anti-human chorionic gonadotrophin antibody (W14A) conjugated to the enzyme, CPG2 (1,000 U kg-1) . Tumour was the only tissue that activated all the prodrug reaching the site . It contained the highest concentration of targeted enzyme conjugate capable of catalysing the reaction of prodrug to drug . Plasma and other tissues were also capable of activating the prodrug but active drug production was limited by the amount of enzyme present . The active drug measured in plasma and tissues other than tumour was attributable to residual antibody-enzyme conjugate at non-tumour sites . Low levels of conjugate in tissues and plasma militate against the advantage of tumour localised enzyme therefore necessitating removal of non-localised enzyme.

J Bacteriol, 1990 Dec, 172(12), 6834 - 40
In vitro analysis of polypeptide requirements of multicomponent phenol hydroxylase from Pseudomonas sp . strain CF600; Powlowski J et al.; An in vitro study of the multicomponent phenol hydroxylase from Pseudomonas sp . strain CF600 was performed . Phenol-stimulated oxygen uptake from crude extracts was strictly dependent on the addition of NAD(P)H and Fe2+ to assay mixtures . Five of six polypeptides required for growth on phenol were necessary for in vitro activity . One of the polypeptides was purified to homogeneity and found to be a flavin adenine dinucleotide containing iron-sulfur protein with significant sequence homology, at the amino terminus, to plant-type ferredoxins . This component, as in other oxygenase systems, probably functions to transfer electrons from NAD(P)H to the iron-requiring oxygenase component . Phenol hydroxylase from this organism is thus markedly different from bacterial flavoprotein monooxygenases commonly used for hydroxylation of other phenolic compounds, but bears a number of similarities to multicomponent oxygenase systems for unactivated compounds.

J Bacteriol, 1990 Dec, 172(12), 6826 - 33
Complete nucleotide sequence and polypeptide analysis of multicomponent phenol hydroxylase from Pseudomonas sp . strain CF600; Nordlund I et al.; Pseudomonas sp . strain CF600 metabolizes phenol and some of its methylated derivatives via a plasmid-encoded phenol hydroxylase and meta-cleavage pathway . The genes encoding the multicomponent phenol hydroxylase of this strain are located within a 5.5-kb SacI-NruI fragment . We report the nucleotide sequence and the polypeptide products of this 5.5-kb region . A combination of deletion analysis, expression of subfragments in tac expression vectors, and identification of polypeptide products in maxicells was used to demonstrate that the polypeptides observed are produced from the six open reading frames identified in the sequence . Expression of phenol hydroxylase activity in a laboratory Pseudomonas strain allows growth on phenol, owing to expression of this enzyme and the chromosomally encoded ortho-cleavage pathway . This system, in conjunction with six plasmids that each expressed all but one of the polypeptides, was used to demonstrate that all six polypeptides are required for growth on phenol.

J Bacteriol, 1990 Dec, 172(12), 6818 - 25
Comparative genetic organization of incompatibility group P degradative plasmids; Burlage RS et al.; Plasmids that encode genes for the degradation of recalcitrant compounds are often examined only for characteristics of the degradative pathways and ignore regions that are necessary for plasmid replication, incompatibility, and conjugation . If these characteristics were known, then the mobility of the catabolic genes between species could be predicted and different catabolic pathways might be combined to alter substrate range . Two catabolic plasmids, pSS50 and pSS60, isolated from chlorobiphenyl-degrading strains and a 3-chlorobenzoate-degrading plasmid, pBR60, were compared with the previously described IncP group (Pseudomonas group P-1) plasmids pJP4 and R751 . All three of the former plasmids were also members of the IncP group, although pBR60 is apparently more distantly related . DNA probes specific for known genetic loci were used to determine the order of homologous loci on the plasmids . In all of these plasmids the order is invariant, demonstrating the conservation of this "backbone" region . In addition, all five plasmids display at least some homology with the mercury resistance transposon, Tn501, which has been suggested to be characteristic of the beta subgroup of the IncP plasmids . Plasmids pSS50 and pSS60 have been mapped in detail, and repeat sequences that surround the suspected degradation genes are described.

Biokhimiia, 1990 Dec, 55(12), 2171 - 81
{Comparative study of aromatic ring meta-cleavage enzymes in Pseudomonas strains with plasmid and chromosomal genetic control of the catabolism of biphenyl and m-toluate}; Selifonov SA et al.; It was shown that two different enzymes of aromatic ring oxidative meta-cleavage (2,3-dihydroxybiphenyl-1,2-dioxygenase), DBO and catechol-2,3-dioxygenase, C230) function in Pseudomonas strains with a plasmid and chromosomal genetic control of biphenyl and toluate catabolism . A comparative analysis of DBO's and C230's expressed by the pBS241 biphenyl degradative plasmid in P . putida BS893, pBS311 in P . putida U83, chromosomal genes in P . putida BF and C230 from P . putida PaW160 (pWWO) was carried out . It was found that the DBO's of all strains under study are highly specialized enzymes in respect of 2,3-dihydroxybiphenyl cleavage and are also able to cleave 3-methyl-catechol and catechol (but not 4-methylcatechol) at low rates . In contrast with DBO's, in Pseudomonas strains the substrate specificities of all C230's are variable . The C230's expressed by the D-plasmids pBS241 and pBC311 have a moderate affinity for catechol, 3-methyl- and 4-methylcatechol, but are unable to cleave 2,3-dihydroxybiphenyl . The C230 which is encoded by the chromosomal structure gene from P . putida BF is very similar to C230 which codes for the TOL-plasmid pWWO . These plasmid differ from C230's expressed by biphenyl D-plasmids due to their capability to cleave 2,3-dihydroxybiphenyl in addition to catechol cleavage . All DBO's and C230's under study possess a number of properties that are typical for the enzymes having an oxidative meta-cleaving effect . The different roles of these enzymes in biphenyl and toluate catabolism in Pseudomonas strains are discussed.

Clin Prev Dent, 1990 Dec, 12(5), 10 - 4
Inhibition of dental plaque formation by mouthwash containing an endo-alpha-1, 3 glucanase; Inoue M et al.; The effect of mouthrinsing with the purified endo-alpha-1, 3 glucanase (mutanase) from a Pseudomonas sp . strain on the formation of dental plaque was investigated . Twenty-two college students participated in the clinical trial . After the labial and lingual surfaces of the 12 front teeth had been subjected to a thorough prophylaxis, the participants were instructed to cease all active oral hygiene practices and to rinse their mouths with either the mutanase or placebo mouthwashes for one minute, up to four times daily, in all, eight times for three days . No restriction regarding meals was given during the experimental period . After a 5-day recess, a second experiment was performed using the same procedures, except the placebo and active mouthwashes were switched . The amount of dental plaque formed on the front teeth was scored according to the criteria of Quigley and Hein . Mean plaque scores were significantly lower for the mutanase (p less than 0.05-0.001) than for the placebo group . In the intraparticipant comparison, most plaque scores were also significantly reduced in general (p less than 0.05-0.0010) by rinsing with the mutanase mouthwash . These data indicate that mutanase is able to suppress the accumulation of dental plaque in humans.

Eur J Epidemiol, 1990 Dec, 6(4), 436 - 7
Isolation of Flavimonas oryzihabitans (CDC group Ve-2) from catheter-induced bacteremia in an immunocompromised patient; Mutters R et al.; Bacteria of the newly proposed genus and combination Flavimonas oryzihabitans, previously known as CDC group Ve-2 or Pseudomonas oryzihabitans, are uncommon pathogens . We report here the first isolation of the organism in Germany from a case of bacteremia and describe the phenotypic characteristics of the strain.

Protein Eng, 1990 Dec, 4(2), 113 - 9
The structure of iron superoxide dismutase from Pseudomonas ovalis complexed with the inhibitor azide; Stoddard BL et al.; The 2.9 A resolution structure of iron superoxide dismutase (FeSOD) (EC 1.15.1.1) from Pseudomonas ovalis complexed with the inhibitor azide was solved . Comparison of this structure with free enzyme shows that the inhibitor is bound at the open coordination position of the iron, with a bond length of 2.0 A . The metal moves by 0.4 A into the trigonal plane to produce an orthogonal geometry at the iron . Binding of the inhibitor also causes a movement of the axial ligand (histidine 26) away from the metal, a lengthening of the iron-histidine bond, and a rotation of the histidine 74 ring . The inhibitor possesses contacts in the binding pocket with a pair of conserved tryptophan residues and with the side chains of tyrosine 34 and glutamine 70 . This glutamine is conserved between all FeSODs, but is absent in MnSOD . Comparisons with MnSOD show that a different glutamine which possesses the same interactions in the active site as Gln70 in FeSOD is conserved at position 154 in the overall SOD sequence, implying that while manganese and FeSODs are structural homologues in a global sense, their functional and evolutionary relationship is that of second-site mutation revertants.

Burns, 1990 Dec, 16(6), 426 - 31
Burns caused by the terrorist bombing of the department store Hipercor in Barcelona . Part 2; Jimenez-Hernandez FH et al.; The Zawacki method was selected for the evaluation of the 20 patients admitted to our Burn Centre after the terrorist attack on 18 June 1987 . The group comprised seven men and 13 women whose ages ranged from 25 to 66 years . The TBSA burn averaged 53 per cent and FTSA burn averaged 32.65 per cent; 25 per cent had respiratory lesions and 10 per cent reported previous bronchopulmonary pathology . The definitive assessment of mortality probability was made between 18 and 24 h after the accident . Of the group, 30 per cent died . All except one had a mortality probability of 100 per cent, 10 per cent of these patients survived . In general, complications appeared during the first 3 weeks; among the infections there was no predominant bacterial species and we had no pseudomonas sepsis cases; respiratory infections were the most important and serious and the most common cause of death . Prophylactic treatment was a determining factor for improving survival . The predominant sequelae were hypertrophic scars and 64 per cent of survivors still suffer with functional sequelae, however 78 per cent of the group have returned to their normal work.

Nucleic Acids Res, 1990 Nov 25, 18(22), 6673 - 6
A single oligonucleotide can be used to rapidly isolate DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction; Rich JJ et al.; We have developed a strategy to rapidly construct DNA hybridization probes for the isolation of genes disrupted by transposon Tn5 insertions . A single oligonucleotide complementary to and extending outward from the ends of the inverted repeat of Tn5 was used to prime DNA synthesis in the polymerase chain reaction . The amplified product consisted of DNA sequences adjacent to both ends of the transposon insertion . The general feasibility of the approach was tested by amplifying pBR322 sequences from a derivative of pBR322 containing a Tn5 insertion . To amplify genomic DNA sequences flanking a Tn5 insertion in the chromosome of a Pseudomonas syringae strain, circular substrates were generated by ligating EcoRI-digested genomic DNA . Tn5 was contained intact within one such circular molecule, as the transposon does not contain sites for cleavage by EcoRI . The amplified product (approximately 2.5 kb) was used as a DNA hybridization probe to isolate the homologous fragment from a cosmid library of wild-type Pseudomonas syringae genomic DNA . This approach may be applied to the efficient isolation of sequences flanking any Tn5 insertion.

J Biol Chem, 1990 Nov 25, 265(33), 20678 - 85
Processing of Pseudomonas exotoxin by a cellular protease results in the generation of a 37,000-Da toxin fragment that is translocated to the cytosol; Ogata M et al.; Pseudomonas exotoxin (PE) was incubated with cells and extracts analyzed for processed fragments . PE was proteolytically cleaved to produce a N-terminal 28-kDa and a C-terminal 37-kDa fragment, the latter being composed of a portion of domain II and all of domain III (the ADP-ribosylating domain) . Cleavage was evident at 10 min after toxin addition and endosome preparations contained the processed fragments . Initially, the two fragments were linked by a disulfide bond . Subsequently, the 37-kDa fragment was reduced and translocated to the cytosol where it inactivated protein synthesis . Cytosol from toxin-treated cells was greatly enriched in the 37-kDa fragment . The 37-kDa fragment appears to be essential for toxicity since mutant PE molecules that do not produce this fragment, or cannot deliver it to the cytosol, fail to kill cells.

Lancet, 1990 Nov 3, 336(8723), 1094 - 6
Person-to-person transmission of Pseudomonas cepacia between patients with cystic fibrosis; LiPuma JJ et al.; Ribotyping, a method of strain identification based on analysis of bacterial genomic restriction fragment length polymorphisms, was used to investigate the acquisition of Pseudomonas cepacia by a patient with cystic fibrosis . Analysis of isolates recovered from the index patient and his contacts showed person-to-person transmission of this opportunist organism . This documentation of the transmission of P cepacia from one cystic fibrosis patient to another suggests that measures to limit the acquisition of the pathogen by patients with cystic fibrosis may be worth while.

Br J Clin Pract, 1990 Nov, 44(11), 512 - 3
Hazards of ear-piercing procedures which traverse cartilage: a report of Pseudomonas perichondritis and review of other complications; Cumberworth VL et al.; A case of severe Pseudomonas perichondritis following a 'fashionable' ear-piercing procedure, performed high on the pinna, is reported . The current vogue for such 'high' ear-piercing, which traverses cartilage rather than the fatty tissue of the ear lobe as in 'traditional' ear-piercing, increases the risk of infection which may produce severe cosmetic deformity.

J Appl Bacteriol, 1990 Nov, 69(5), 750 - 7
Alterations in the major heterotrophic bacterial populations isolated from a still bottled mineral water; Morais PV et al.; The heterotrophic bacterial population of a bottled mineral water stored in returnable glass bottles and in polyvinyl chloride (PVC) bottles at room temperature was studied over 9-12 months . The plate counts in R2A medium incubated at 22 degrees and 37 degrees C were low initially, increasing to 10(4)-10(5) cfu/ml within a few days of bottling . The number of bacteria recovered at 22 degrees C from PVC bottles was fairly constant during the storage period, but the population isolated at 37 degrees C decreased markedly after storage for 1 year . The major components of the population were Pseudomonas strains, one of which was identified as Pseudomonas vesicularis . Major changes took place during storage; two groups of bacteria (B and C) were dominant initially, but during the latter period of storage other groups (F, G and H) increased in number.

Appl Environ Microbiol, 1990 Nov, 56(11), 3598 - 600
Prolonged survival of Pseudomonas cepacia in commercially manufactured povidone-iodine; Anderson RL et al.; Pseudomonas cepacia organisms were recently recovered from a povidone-iodine antiseptic solution . During the subsequent investigation, laboratory studies were initiated to determine the survival time of these organisms in the iodophor solution, which contains 1% titratable iodine . The solution was sampled weekly upon receipt in our laboratory, and P . cepacia was subsequently recovered through 29 weeks of sampling . Current laboratory data and lot production date information from the manufacturer indicate that P . cepacia survived for up to 68 weeks from the time of manufacture . Scanning electron microscopic examination of contaminated solution demonstrated bacterial cells embedded in extracellular material.

Appl Environ Microbiol, 1990 Nov, 56(11), 3382 - 8
Cloning of a gene from Pseudomonas sp . strain PG2982 conferring increased glyphosate resistance; Fitzgibbon JE et al.; A plasmid carrying a 2.4-kilobase-pair fragment of DNA from Pseudomonas sp . strain PG2982 has been isolated which was able to increase the glyphosate resistance of Escherichia coli cells . The increase in resistance was dependent on the presence of a plasmid-encoded protein with a molecular weight of approximately 33,000, the product of a translational fusion between a gene on the vector, pACYC184, and the insert DNA . An overlapping region of the PG2982 chromosome carrying the entire gene (designated igrA) was cloned, and a plasmid (pPG18) carrying the gene was also able to increase glyphosate resistance in E . coli . A protein with a molecular weight of approximately 40,000 was encoded by the PG2982 DNA contained in pPG18 . This plasmid was not able to complement a mutation in the gene for 5-enolpyruvylshikimate-3-phosphate synthase (aroA) in E . coli, and modification of glyphosate by E . coli cells containing the plasmid could not be demonstrated . The nucleotide sequence of the PG2982 DNA contained an open reading frame able to encode a protein with a calculated molecular weight of 39,396.

Toxicol Lett, 1990 Nov, 54(1), 39 - 46
Perfluoro-N-decanoic acid effects on enzymes of fatty acid metabolism; Singer SS et al.; In vitro perfluorodecanoate (PFDA) effects on Pseudomonas acyl-CoA synthetase, Candida acyl-CoA oxidase and pigeon muscle carnitine acetyltransferase were examined . Synthetase made little PFDA-CoA from PFDA . It used palmitate, oleate, laurate and decanoate more extensively . PFDA inhibited acyl-CoA formation from these acids . Palmitoyl-CoA formation was affected most . That of decanoyl-CoA was affected least . Inhibitions appeared to be competitive . Acyl-CoA oxidase test substrates were palmitoyl-CoA, lauroyl-CoA and decanoyl-CoA . Oxidase preferred C-10 and C-12 acyl-CoAs . PFDA inhibited oxidation of C-10 and C-12 acyl-CoAs more than that of palmitoyl-CoA . Inhibitions with C-16 and C-10 acyl-CoAs were competitive, KIs 593 +/- 150 and 76 +/- 6.0 microM . Acetyl-CoA was the best acetyltransferase substrate . C-2 to C-8 transfer from acyl-CoAs was inhibited similarly by PFDA . Inhibitions of C-2 and C-8 transfer were competitive and non-competitive, respectively, KIs 111 +/- 15 and 76 +/- 28 microM.

J Clin Invest, 1990 Nov, 86(5), 1684 - 9
CD4-Pseudomonas exotoxin conjugates delay but do not fully inhibit human immunodeficiency virus replication in lymphocytes in vitro; Tsubota H et al.; The CD4 molecule is a high affinity receptor for the human immunodeficiency virus (HIV) envelope glycoprotein (gp160 or gp120) . This glycoprotein is expressed on the surface membrane of cells infected with HIV . It has, therefore, been suggested that a soluble form of CD4 might be used as a targeting agent to deliver toxins selectively to cells infected with HIV . We demonstrate that CD4-Pseudomonas exotoxin A (PE) conjugates inhibit the proliferation of gp160-transfected Chinese hamster ovary cells and block HIV replication in virus-infected H9 cells . However, this inhibition of HIV replication appears to be incomplete since virus replication occurs following removal of the toxin conjugates from these cultures . Moreover, CD4-PE conjugates delay but do not inhibit HIV replication in human peripheral blood lymphocytes . These studies suggest that such conjugates should be assessed only as potential adjunctive therapies in the acquired immunodeficiency syndrome.

Proc Natl Acad Sci U S A, 1990 Nov, 87(21), 8291 - 5
The recombinant immunotoxin anti-Tac(Fv)-Pseudomonas exotoxin 40 is cytotoxic toward peripheral blood malignant cells from patients with adult T-cell leukemia; Kreitman RJ et al.; Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin containing the heavy and light variable regions of the anti-Tac monoclonal antibody fused to a mutant form of Pseudomonas exotoxin (PE) . Anti-Tac binds to the p55 subunit of the human interleukin 2 (IL-2) receptor, and anti-Tac(Fv)-PE40 kills human or monkey cell lines that contain either the intact IL-2 receptor or its p55 subunit alone . To assess the usefulness of anti-Tac(Fv)-PE40 in treatment of IL-2 receptor-positive leukemia, we tested peripheral blood mononuclear cells from six patients with adult T-cell leukemia . In each of the six patients, anti-Tac(Fv)-PE40 was extremely cytotoxic to the malignant cells . Metabolic activity and sensitivity of the fresh cells improved when a small amount of IL-2 (10 units per ml) was present during incubation . The toxin concentration necessary to inhibit protein synthesis by 50% after 16-hr incubation of cells with immunotoxin varied from 1.6 to 16 ng/ml (2.5-25 x 10(-11) M) . In every case, binding was by means of the Tac antigen because anti-Tac(Fv)-PE40 cytotoxicity was prevented by adding excess anti-Tac antibody . Moreover, anti-Tac alone or an inactive mutant of anti-Tac(Fv)-PE40 without ADP-ribosylation activity had very little cytotoxic activity . Peripheral blood mononuclear cells from normal controls, from a patient with Tac-negative leukemia, and from adult T-cell leukemia patients without significant peripheral blood involvement were not sensitive to anti-Tac(Fv)-PE40 . These results indicate that anti-Tac(Fv)-PE40 is a potent cytotoxin against adult T-cell leukemia cells in vitro and warrants clinical testing.

J Clin Immunol, 1990 Nov, 10(6 Suppl), 19S - 28S; discussion 28S-29S
Lymphokine receptor-directed therapy: a model of immune intervention; Waldmann TA et al.; We have proposed a multichain model for the high-affinity interleukin-2 (IL-2) receptor involving two IL-2-binding peptides, a 70/75 kilodalton (kD) and a 55 kD, reactive with the anti-Tac monoclonal antibody, which are associated in a receptor complex . With the use of coprecipitation analysis, radiolabeled interleukin-2 cross-linking procedures, and flow cytometric resonance energy transfer measurements, a series of additional peptides of molecular weight 22,000, 35,000, 40,000, 75,000 (non-IL-2 binding), 95,000-105,000, and 180,000 has been associated with the two interleukin-2-binding peptides . In contrast to resting T cells, the abnormal T cells of patients with human T-cell lymphotropic virus I-associated adult T-cell leukemia, patients with select autoimmune disorders, and individuals rejecting allografts express the Tac peptide (p55) of the IL-2 receptor . To exploit this difference in Tac antigen expression, we have initiated therapeutic trials using unmodified anti-Tac, conjugates of anti-Tac with truncated Pseudomonas exotoxin PE-40, interleukin-2-truncated toxin fusion proteins, and alpha- and beta-emitting isotopic chelates of anti-Tac . Furthermore, by genetic engineering humanized hyperchimeric anti-Tac molecules have been prepared in which the molecule is entirely human IgG1, except for the small complementarity-determining regions that are retained from the mouse antibody . This "humanized" antibody manifested the ability to perform antibody-dependent cellular cytotoxicity absent in the original mouse monoclonal . The clinical application of anti-interleukin-2 receptor-directed therapy represents a new perspective for the treatment of certain neoplastic diseases and autoimmune disorders and for the prevention of allograft rejection.

J Gen Microbiol, 1990 Nov, 136 ( Pt 11), 2307 - 17
Comparative studies on the degradation of guanidino and ureido compounds by Pseudomonas; Tricot C et al.; The utilization of guanidino and ureido compounds was studied in several Pseudomonas species . Multiple routes of agmatine catabolism were found . All members of the homology group I of Pseudomonas use the initial deamination of agmatine to carbamoylputrescine which is subsequently converted to putrescine . In Pseudomonas indigofera, the catabolism of agmatine can also occur via an initial hydrolysis of the amidino group to putrescine catalyzed by an agmatine amidinohydrolase . A third pathway was found in Pseudomonas cepacia, namely oxidative deamination producing guanidinobutyraldehyde catalyzed by agmatine dehydrogenase, followed by formation of guanidinobutyrate and removal of urea by guanidinobutyrate amidinohydrolase to produce 4-aminobutyrate . Novel amidino-hydrolases were characterized in P . putida for the utilization of arcaine and audouine, and in P . cepacia for arcaine, homoarginine and guanidinovalerate . Guanidinovalerate amidinohydrolase was also detected in P . doudoroffii . Some of these amidinohydrolases accept more than one substrate, e.g., guanidinobutyrate and guanidinovalerate utilization by P . doudoroffii and P . cepacia, the catabolism of arcaine and audouine by P . putida, and the degradation of arcaine and homoarginine by P . cepacia.

J Immunol, 1990 Oct 15, 145(8), 2766 - 71
IL-2-PE40 prevents the development of tumors in mice injected with IL-2 receptor expressing EL4 transfectant tumor cells; Kozak RW et al.; A number of different immunotherapeutic reagents are currently being developed to target IL-2R for the treatment of leukemia, graft rejection, and certain autoimmune diseases . Previously, we have shown that IL-2-PE40, a chimeric protein composed of human IL-2 linked to the N-terminus of a truncated form of Pseudomonas exotoxin (PE), could effectively kill a variety of cell lines in vitro expressing either low, intermediate, or high affinity IL-2R . Here, we demonstrate that IL-2-PE40 can successfully retard or prevent the growth of a lethal ascites tumor or a solid tumor composed of EL4J murine thymoma cells transfected with the p55 murine IL-2R . The transfected line, EL4J-3.4, expresses 1,000 to 3,000 high affinity IL-2R . Survival extension in the ascites model was achieved by initiating treatment either after 4 to 6 h or within 5 days post-tumor injection in both athymic nude and C57BL/6 mice . Similarly, the growth of an aggressive s.c . solid tumor could also be inhibited . Extension of survival was not achieved either by using the truncated toxin alone not attached to IL-2 or by using an IL-2-PE40Asp553 mutant lacking a functional toxin . Survival extension was not caused by IL-2 activated NK or other host effector mechanisms as IL-2-PE40 was unable to prevent the receptor-negative EL4J parental line from forming a lethal ascites or a solid tumor . Thus, IL-2-PE40 is a potent, specific cytolytic reagent that may prove useful in the arsenal of anti-IL-2R immunotherapeutics.

Can J Microbiol, 1990 Oct, 36(10), 676 - 81
Preliminary study on relationships among strains forming a bacterial community selected on naphthalene from a marine sediment; Tagger S et al.; Two bacterial strains were isolated from a bacterial community formed of nine strains, selected from a marine sediment on a seawater medium with naphthalene as sole carbon source . The two strains studied in the present work were the only strains of this community able to grow in pure culture on naphthalene; therefore, they were called "primary" strains . The seven other strains were maintained in the community by using metabolic intermediates of the two primary strains; they were called "auxiliary" strains . Regulation of naphthalene metabolism was studied for the two primary strains . They oxidized naphthalene into catechol, which was degraded only by the meta pathway . For Pseudomonas Lav . 4, naphthalene oxygenase and salicylate hydroxylase were inducible; catechol 2,3-dioxygenase was constitutive . For Moraxella Lav . 7, naphthalene oxygenase was constitutive; salicylate hydroxylase and catechol 2,3-oxygenase were inducible . The Moraxella strain carries two cryptic plasmids, about 63- and 85-kb in molecular size . In the bacterial community culture medium, Moraxella Lav . 7 prevented accumulation of 2-hydroxymuconate semialdehyde formed by Pseudomonas Lav . 4 . The auxiliary strains take up formic, acetic, pyruvic, propionic, and succinic acids released by the two primary strains.

Wei Sheng Wu Xue Bao, 1990 Oct, 30(5), 393 - 6
{Classify species of Pseudomonas pseudomallei into serotypes}; Han O et al.; Species of P . pseudomallei can be classified into two serotypes, serotype I and serotype II, based on whether or not it contains a thermolabile antigen beside a thermostable one . Under the condition of lack of typing serum, by means of serum absorption test, we recognized a strain which contains a major thermolabile antigen . The antigen was purified by Sephadex G-200, and it was used to inoculate rabbits . With the immunoserum at hand, we identified 68 of the domestic chinese strains and 6 of alien strains for serotyping by bi-directional agar diffusion test . The results showed that 68 strains were identified serotype I, 3 strains serotype II, and the remaining 3 comparable with those reported indicating that strains of serotype I were found mostly in Asia, and that the serotype are unrelated to their existing environments, nor that of animal bodies, but connected with their existence in geographic distribution.

Vet Microbiol, 1990 Oct, 25(1), 77 - 85
Development of an avidin-biotin dot enzyme-linked immunosorbent assay and its comparison with other serological tests for diagnosis of glanders in equines; Verma RD et al.; A dot enzyme-linked immunosorbent assay (dot ELISA) was developed for diagnosis of glanders in equines . The test was based on the detection of IgG antibodies to Pseudomonas mallei antigens bound to nitrocellulose coated on plastic strips (dipsticks), the reaction being amplified by an avidin-biotin system with biotinylated anti-horse IgG and horseradish peroxidase-avidin D . Sera from 810 normal, six naturally infected and 48 sensitized equines were tested by this assay, and results were compared with complement fixation, indirect haemagglutination and counter-immunoelectrophoresis tests . Dot ELISA had the highest sensitivity, and was superior to other tests in that it was rapid and easy to perform, the results were easy to interpret, the assay was not influenced by anti-complement activity, and it was able to detect antibodies at an early stage . Testing of serum at 1:200 dilution is proposed for epidemiological screening.

J Med Microbiol, 1990 Oct, 33(2), 115 - 20
Pathogenic factors of Pseudomonas cepacia isolates from patients with cystic fibrosis; Gessner AR et al.; One hundred and nineteen isolates of Pseudomonas cepacia, 98 of which were from cystic fibrosis (CF) patients and 21 from environmental and other human sources, were examined for biochemical and exo-enzymatic properties that may contribute to the pathogenicity of this bacterium . The following characteristics were demonstrated significantly more frequently in isolates from CF patients than in control isolates: production of catalase, ornithine decarboxylase, valine aminopeptidase, C14 lipase, alginase and trypsin; reduction of nitrate to nitrite; hydrolysis of urea and xanthine; complete haemolysis on bovine red blood cells; cold-sensitive haemolysis on human red blood cells; greening of horse and rabbit red blood cells . The role of these factors in the pulmonary disease associated with cystic fibrosis is not clear . However, several factors which have been reported previously as being associated with pathogenic processes with other bacteria have now been described in P . cepacia . Additional factors not previously reported as "pathogenicity factors" are also described.

J Clin Microbiol, 1990 Oct, 28(10), 2331 - 4
Production of hemolysin and other extracellular enzymes by clinical isolates of Pseudomonas pseudomallei; Ashdown LR et al.; One hundred clinical isolates of Pseudomonas pseudomallei from humans were tested for their ability to produce extracellular, biologically active substances which are thought to contribute to the virulence of Pseudomonas species . All isolates produced at least on extracellular enzyme; 91 strains were positive for lecithinase, lipase, and protease; but none was positive for elastase . Ninety-three strains produced a hemolysin which was detectable around the heavy growth on saline-washed sheep erythrocyte brain heart infusion agar but not demonstrable around individual colonies or in broth culture filtrate . In contrast, a hemolysin which was cytolytic around individual colonies of P . pseudomallei on the assay plate and in broth culture filtrate was exhibited by four strains . By using one of these four isolates as the test strain, the latter hemolysin was characterized further . It was heat labile, most active in an acid environment (pH 5.5), and cytolytic in broth culture filtrate for a variety of animal and human erythrocytes . Sterols, particularly cholesterol and 7-dehydrocholesterol, inhibited its hemolytic activity, but the activity was not enhanced by reducing agents or suppressed by reagents which modify sulfhydryl-activated hemolysins . A nonhemolytic mutant of the test strain of P . pseudomallei retained the extracellular enzymes of its parent, indicating that the hemolysin was not a lecithinase, lipase, or protease.

Virology, 1990 Oct, 178(2), 364 - 72
Purified phi 6 nucleocapsids are capable of productive infection of host cells with partially disrupted outer membranes; Ojala PM et al.; Purified nucleocapsids of bacteriophage phi 6, lacking the phage lipid envelope, are unable to infect intact Pseudomonas syringae host cells . A method for studying the process by which a naked virus particle, the phi 6 nucleocapsid, penetrates the host cytoplasmic membrane was developed . Host cells were rendered competent for nucleocapsid infection by treatment with repeated washings with salt and sucrose and the subsequent addition of lysozyme . This treatment disrupts the outer membrane, permitting the nucleocapsid to reach the cytoplasmic membrane and to infect the cell . The nucleocapsid infection is blocked by monoclonal antibodies raised against the nucleocapsid shell protein P8.

Laryngoscope, 1990 Oct, 100(10 Pt 1), 1112 - 5
Gentamicin iontophoresis in the treatment of bacterial otitis externa in the guinea pig model; King DM 3rd et al.; Pseudomonas otitis externa is one of the most common infections treated by otolaryngologists . Infections induced in 30 guinea pigs appeared similar to that seen in humans . The ears were then placed into four treatment groups: group A, which received a single cleaning; group B, which received a single cleaning followed by gentamicin drops 4 times daily; group C, which received a single cleaning followed by a single gentamicin iontophoresis treatment; and group D, the control group, which received no treatment . Infections were analyzed by grading edema, purulence, and erythema . An average of 10.2 days was required for control group to return to normal appearance . Groups A, B, and C had mean resolution times of 5.9, 4.7, and 4.3 days, respectively . Gentamicin iontophoresis appears to be promising, with results as good as drop therapy in otitis externa in the guinea pig model.

J Bacteriol, 1990 Oct, 172(10), 5774 - 82
In vitro replication, packaging, and transcription of the segmented double-stranded RNA genome of bacteriophage phi 6: studies with procapsids assembled from plasmid-encoded proteins; Gottlieb P et al.; The genome of the lipid-containing bacteriophage phi 6 contains three segments of double-stranded RNA (dsRNA) . We prepared cDNA copies of the viral genome and cloned this material in plasmids that replicate in Escherichia coli and Pseudomonas phaseolicola, the natural host of phi 6 . These plasmids direct the formation of viral proteins and the assembly of structures similar to viral procapsids containing proteins P1, P2, P4, and P7 . We found that these particles are capable of taking up viral single-stranded RNA and synthesizing the minus strands to produce dsRNA structures . Once the dsRNA is formed, it is then used as a template for the production of viral plus strands in a reaction that resembles normal transcription . The particles were also capable of directly transcribing exogenous dsRNA . The replicase reactions were specific for phi 6 RNA, were specific for procapsids, and resulted in substantial incorporation of product dsRNA into particles . These results offer strong support to a model in which genomic packaging is done by preformed procapsids.

Proc Natl Acad Sci U S A, 1990 Oct, 87(20), 8155 - 9
Transfer and expression of an organophosphate insecticide-degrading gene from Pseudomonas in Drosophila melanogaster; Phillips JP et al.; The organophosphorus acid hydrolases represent a distinct class of enzymes that catalyze the hydrolysis of a variety of organophosphate substrates, including many insecticides and their structural analogues . The plasmid-borne opd gene of Pseudomonas diminuta strain MG specifies an organophosphorus acid hydrolase, a phosphotriesterase, that has been well characterized and can hydrolyze a broad spectrum of insect and mammalian neurotoxins . The in situ functioning of this enzyme in the metabolism of organophosphates has been analyzed directly in insects by transferring the opd gene into embryos of Drosophila melanogaster by P element-mediated transformation . The chromosomal locations of this stably inherited transgenic locus differed from strain to strain and demonstrated various expressivity on the whole-insect basis . Transcriptional induction of opd in one of these strains under control of the Drosophila heat shock promoter, hsp70, resulted in the synthesis of stable active enzyme that accumulated to high levels with repeated induction . The heat shock-induced synthesis of organophosphorus acid hydrolases in transgenic flies conferred enhanced resistance to toxic paralysis by the organophosphate insecticide paraoxon.

Cancer Res, 1990 Oct 1, 50(19), 6379 - 88
Enhanced therapeutic efficacy against an ovarian tumor xenograft of immunotoxins used in conjunction with recombinant alpha-interferon; Pearson JW et al.; The antitumor effects of two immunotoxins were evaluated in vitro and in vivo against the human ovarian carcinoma cell line, OVCAR-3 . The immunotoxins used were composed of recombinant ricin A chain (rRTA) covalently attached to a monoclonal antibody directed toward the human transferrin receptor (45412/rRTA, also called 454A12 MAB-rRTA by Cetus Corporation) or Pseudomonas exotoxin coupled to an anticarcinoma monoclonal antibody (NR-LU-10/PE) . Preliminary characterization of the NR-LU-10 antigen by immunoprecipitation and cellular fluorescence demonstrated two dominant cell surface polypeptide moieties with molecular weights of 40,000 and 45,000 and a minor component with a molecular weight of 33,000 . The immunotoxins were used alone or in combination with recombinant human alpha-interferon (rhIFN-alpha) . Protein synthesis was inhibited in a dose-dependent manner in OVCA-3 cells incubated in vitro with either NR-LU-10/PE or 454A12/rRTA (50% inhibitory concentrations, 1 and 75 ng/ml, respectively) . Unconjugated NR-LU-10 or 454A12 abrogated the activity of the relevant immunotoxins . Concomitant incubation in vitro of OVCAR-3 cells with NR-LU-10/PE or 454A12/rRTA and a noncytotoxic concentration of rhIFN-alpha potentiated the inhibitory activity of the immunotoxins via a mechanism independent of antigenic upregulation . This potentially synergistic combination was then tested in vivo . The median survival time (MST) of mice given injections i.p . of 4 x 10(6) OVCAR-3 cells was 46 days . Cohorts of mice that received intracavitary treatment beginning 5 days posttumor cell inoculation with either 0.25 or 0.5 microgram of NR-LU-10/PE every other day for a total of 10 treatments exhibited a significantly increased MST of 63 and 104 days, respectively (P less than 0.0001) . Likewise, the i.p . injection of either 2.5 or 10 micrograms of 454A12/rRTA given in an identical schedule resulted in a MST of 89 and greater than 120 days, respectively (P less than 0.0001) . When rhIFN-alpha was administered i.p . in conjunction with those doses of either immunotoxin, a significant increase in the MST was observed in comparison with mice given immunotoxin alone . The combination of 5 x 10(4) units of rhIFN-alpha and 0.25 microgram of NR-LU-10/PE resulted in 67% long-term survivors (greater than 120 days) compared with only 13% survival of mice given the immunotoxin alone . Similarly, 2.5 micrograms of 454A12/rRTA plus rhIFN-alpha resulted in an enhanced therapeutic response (89% long-term survivors) when compared with 454A12/rRTA alone (29%).(ABSTRACT TRUNCATED AT 400 WORDS)

Semin Cancer Biol, 1990 Oct, 1(5), 345 - 50
Selective killing of tumor cells using EGF or TGF alpha-Pseudomonas exotoxin chimeric molecules; Siegall CB et al.; Many types of cancer cells display aberrantly high numbers of EGF receptors on their surface . We have targeted these cells for elimination by combining the cell binding ability of either epidermal growth factor or transforming growth factor type alpha with the potent cell killing activity of Pseudomonas exotoxin . These chimeric molecules are formed either by chemical conjugation of the two proteins or by expression of a gene fusion into a product containing both proteins . In this review, we show that these chimeric toxins are extremely cytotoxic to a variety of cancer cell lines.

Virology, 1990 Oct, 178(2), 509 - 19
RNA-protein complexes responsible for replication and transcription of the double-stranded RNA bacteriophage phi 6; Ewen ME et al.; RNA-protein complexes active for transcription and replication of the double-stranded RNA bacteriophage phi 6 have been partially purified from lysates of infected Pseudomonas phaseolicola . Transcribing particles (filled procapsids) contain the three viral dsRNAs and all four procapsid proteins P1, P2, P4, and P7 . Particles with replicase activity contain the same four proteins as well as single plus RNA strands duplexed with various extents of minus strands initiated in vivo . The in vitro replication reaction is insensitive to RNaseA . Sarkosyl destroys transcription complexes but does not reduce the activity of replication complexes, although the latter lose 80% of their P4 and the single-strand RNA template becomes sensitive to RNase . The detection of complexes that replicate small only, or both small and medium, RNA suggests that the RNAs are packaged sequentially in the order small, medium, large.

J Bacteriol, 1990 Oct, 172(10), 5593 - 601
Indoleacetic acid operon of Pseudomonas syringae subsp . savastanoi: transcription analysis and promoter identification; Gaffney TD et al.; Expression of the indoleacetic acid (iaa) operon, which contributes to the virulence of the phytopathogenic bacterium Pseudomonas syringae subsp . savastanoi, was monitored by using broad-host-range lacZ reporter gene plasmids . A combination of translational (gene) fusions and transcriptional (operon) fusions of P . syringae subsp . savastanoi sequences to lacZ allowed localization of the iaa operon promoter . RNA recovered from P . syringae subsp . savastanoi strains was mapped with iaa operon-specific probes to precisely locate the transcription initiation site . When transcripts from an iaaM::lacZ fusion in Escherichia coli were analyzed, an identical transcription initiation site was observed . The DNA sequence of the iaa operon promoter closely resembled the consensus E . coli promoter sequence . We detected an active, constitutive level of indoleacetic acid biosynthetic gene expression during bacterial growth under a variety of conditions in the absence of host plant influence.

Zh Mikrobiol Epidemiol Immunobiol, 1990 Oct, (10), 41 - 5
{The morphological characteristics of mucoid variants of Pseudomonas pseudomallei}; Kapliev VI et al.; Two types of mucoid colonies formed by P . pseudomallei cultivated on solid culture media have been studied by the method of electron cytochemistry . The intercellular material of primary mucoid colonies has been found to positively react with ruthenium red, which is indicative of its polysaccharide nature . Slime is the product secreted by P . pseudomallei and participates in the formation of the pseudocapsule and colonies . The intercellular material seems to be a sum of biopolymers based on the products of destroyed bacterial cells.

Biochemistry, 1990 Sep 25, 29(38), 8885 - 93
The 2.1-A resolution structure of iron superoxide dismutase from Pseudomonas ovalis; Stoddard BL et al.; The 2.1-A resolution crystal structure of native uncomplexed iron superoxide dismutase (EC 1.15.1.1) from Pseudomonas ovalis was solved and refined to a final R factor of 24% . The dimeric structure contains one catalytic iron center per monomer with an asymmetric trigonal-bipyramidal coordination of protein ligands to the metal . Each monomer contains two domains, with the trigonal ligands (histidines 74 and 160; aspartate 156) contributed by the large domain and stabilized by an extended hydrogen-bonded network, including residues from opposing monomers . The axial ligand (histidine 26) is found on the small domain and does not participate extensively in the stabilizing H-bond network . The open axial coordination position of the iron is devoid of bound water molecules or anions . The metal is located 0.5 A out of the plane of the trigonal ligands toward histidine 26, providing a slightly skewed coordination away from the iron binding site . The molecule contains a glutamine residue in the active site which is conserved between all iron enzymes sequenced to data but which is conserved among all manganese SODs at a separate position in the sequence . This residue shows the same structural interactions in both cases, implying that iron and manganese SODs are second-site revertants of one another.

J Biol Chem, 1990 Sep 25, 265(27), 16318 - 23
Cytotoxicity of IL6-PE40 and derivatives on tumor cells expressing a range of interleukin 6 receptor levels; Siegall CB et al.; The chimeric toxin IL6-PE40, which is composed of interleukin 6 (IL6) fused to a mutant form of Pseudomonas exotoxin (PE) devoid of its native cell recognition domain, can kill myeloma and hepatoma cells which express high levels of IL6 receptors . To enhance the usefulness of IL6-PE40 on potential target cells, we have attempted to develop more potent IL6-PE derivatives . We have developed nine new IL6-PE derivatives and assessed their cytotoxicity on human myeloma cells . Two of these new forms, IL6-domain II-PE40 and IL6-PE664Glu were more toxic to myeloma cells bearing IL6 receptors than was IL6-PE40 . These two chimeric toxins were compared with IL6-PE40 for cytotoxicity toward a variety of tumor cell lines . We found that most tumor cell lines which are sensitive to IL6-PE40 are more sensitive to IL6-domain II-PE40 and IL6-PE664Glu . Cells with as few as 200-600 IL6 receptors/cell could be killed . The specificity of these chimeric toxins was shown through competition with recombinant IL6 . Toxicity studies in mice demonstrated that the two new molecules had an LD50 of 10-20 micrograms/mouse . This compares to an IL6-PE40 LD50 of 20 micrograms/mouse . The new IL6-toxins could be detected in the serum up to 8 h after intraperitoneal administration with a peak at 1 h . These data suggest that IL6-domain II-PE40 and IL6-PE664Glu may be more useful than IL6-PE40 in killing IL6 receptor-bearing tumor cells in animals.

J Biol Chem, 1990 Sep 25, 265(27), 16306 - 10
Mutagenesis of Pseudomonas exotoxin in identification of sequences responsible for the animal toxicity; Chaudhary VK et al.; Pseudomonas exotoxin (PE) is composed of three structural domains that are responsible for cell recognition, membrane translocation, and ADP-ribosylation . The deletion of the cell recognition domain (domain Ia) of PE results in a molecule that does not bind to target cells and has low toxicity in mice (Hwang, J., FitzGerald, D.J.P., Adhya, S., and Pastan, I . (1987) Cell 48, 129-136) . To determine the specific sequences required for cell binding as well as cell and animal toxicity, a series of domain I mutants was constructed . Using a T7 promoter-based expression system and an OmpA signal sequence, large amounts of the various mutant toxins were secreted into the periplasm from which they were easily purified in milligram quantities . The data indicate that amino acids at positions 246, 247, and 249 have an important role in the toxicity of PE . Conversion of these amino acids to glutamic acid or glycine but not to lysine or deletion of amino acids 241-250 diminishes the toxicity of PE . When combined with a mutation at position 57 a molecule is created that has very low toxicity against cultured cells or in mice.

J Biol Chem, 1990 Sep 25, 265(27), 16311 - 7
IL2-PE664Glu, a new chimeric protein cytotoxic to human-activated T lymphocytes; Lorberboum-Galski H et al.; To produce a molecule that will kill activated T cells as well as lymphomas and leukemias expressing interleukin 2 (IL2) receptors, we have created a recombinant chimeric protein in which IL2 is attached in peptide linkage to a truncated mutant form of Pseudomonas exotoxin (PE) (Lorberboum-Galski, H., FitzGerald, D.J.P., Chandhary, V.K., Adhya, S., and Pastan, I . (1988) Proc . Natl . Acad . Sci . U.S.A . 85, 1922-1926) . Although this molecule was very active on rodent cells, it had lower activity on some human cell types . A new chimeric protein termed IL2-PE664Glu has been constructed that is extremely toxic to both phytohemagglutinin blasts and mixed leukocyte reaction blasts prepared from monkey and human lymphocytes . The chimeric gene encoding this protein was constructed by fusing a cDNA clone for human interleukin 2 to the 5' end of a mutated cDNA encoding a full-length PE molecule . Four amino acids in domain I of PE were changed thus decreasing its nonspecific toxicity . IL2-PE664Glu is a much more active cytotoxic molecule for primate and human-activated T cells than IL2-PE40 which is a chimeric protein that was found to be an effective immunosuppressive agent in rodent models . Our results indicate that IL2-PE664Glu should be evaluated as an immunosuppressive agent for the treatment of human immune disorders in which activated T cells expressing the IL2 receptor are prominent.

Cancer Res, 1990 Sep 15, 50(18), 5992 - 6
Selective elimination of breast cancer cells from human bone marrow using an antibody-Pseudomonas exotoxin A conjugate; Bjorn MJ et al.; A pancarcinoma monoclonal antibody (NR-LU-10), homogeneously reactive with human breast cancer cells, was conjugated to Pseudomonas exotoxin A . The immunotoxin was evaluated for its potential for purging breast cancer cells from human bone marrow . The immunotoxin NR-LU-10 antibody did not react with normal bone marrow preparations yet readily detected 1% contamination of bone marrow by MCF-7 breast cancer cells added to normal bone marrow without significantly inhibiting the colony-forming ability of bone marrow progenitor cells . NR-LU-10-Pseudomonas exotoxin A has potential for purging bone marrow of breast cancer cells without impairing the growth of bone marrow progenitor cells.

J Biol Chem, 1990 Sep 5, 265(25), 15198 - 202
Anti-Tac(Fv)-PE40, a single chain antibody Pseudomonas fusion protein directed at interleukin 2 receptor bearing cells; Batra JK et al.; Anti-Tac(Fv)-PE40 is a chimeric single chain immunotoxin in which anti-Tac variable heavy and light chains held together by a peptide linker are attached to PE40, a truncated form of Pseudomonas exotoxin . This molecule was shown to be extremely cytotoxic for interleukin 2 (IL2) receptor bearing cells in tissue culture (Chaudhary, V . K., Queen, C., Junghans, R . P., Waldmann, T . A., FitzGerald, D . J., and Pastan, I . (1989) Nature 339, 394-397) . Here we describe various forms of anti-Tac(Fv)-PE40 protein in which the order of the variable domains of anti-Tac has been switched and also three different types of peptide linkers have been used . All these proteins were purified to near homogeneity and were found to have similar cytotoxic activities against various human cells expressing the p55 subunit of the IL2 receptor . Anti-Tac(Fv)-PE40 was also found to have a very potent suppressive activity against phytohemagglutinin-activated human lymphoblasts and in a human mixed lymphocyte reaction . Anti-Tac(Fv)-PE40 appeared in the blood rapidly in mice after intraperitoneal administration and could be detected in the blood for up to 8 h . Anti-Tac(Fv)-PE40 warrants evaluation as an anti-tumor and immunosuppressive agent in humans.

Mol Microbiol, 1990 Sep, 4(9), 1551 - 6
Promoter-upstream activator sequences are required for expression of the xylS gene and upper-pathway operon on the Pseudomonas TOL plasmid; Holtel A et al.; Stimulation of transcription from the Pseudomonas TOL plasmid xylS gene promoter (Ps) and the upper-pathway operon promoter (Pu) is dependent on the positive regulator protein XylR activated by an effector molecule such as 3-cholorotoluene, and on RpoN, an RNA polymerase sigma factor . Mutational analysis of the Ps and Pu promoters showed that upstream activator sequences located between -110 and -218bp upstream of the main transcription initiation point are required for regulated expression from these promoters . A search for homologous nucleotide sequences in the -110 to -218bp region in Pu and Ps revealed conserved sequences that may act as putative recognition sequences for the XylR protein . Ps and Pu exhibit another well-conserved region at around -50bp, which is homologous to corresponding sites in other RpoN-dependent promoters and may constitute a binding site for integration host factor (IHF).

Zentralbl Veterinarmed B, 1990 Sep, 37(7), 491 - 500
{Comparative studies of the paraspecific immunostimulation (paramunization) by bacterial lysates in bacterial infection models and in the cytotoxicity test}; Wieler L et al.; Bacterial lysates of different bacterial strains (E . coli, B . bronchiseptica, P . haemolytica) were prepared by heating, acid- and alkaline-hydrolysis . Lysates were tested for their immunostimulating effect in bacterial infection models and with chromium 51 test demonstrating spontaneous (natural) cytotoxicity . Lysate production was standardized by protein- and Lps-determination . The alkaline-hydrolysis reduced toxicity of Lps and increased the content of soluble bacterial protein . Heating and acid-hydrolysis did not alter bacterial suspensions with respect to Lps-toxicity and protein-content . Mice infected with P . aeruginosa, P . multocida, E . coli and L . monocytogenes (5-10 LD50) had a significantly longer survival time after prophylactic immunostimulation with bacterial lysates than control animals . No protection was observed in immunostimulated mice infected with Erysipelothrix rhusiopathiae . In the Pseudomonas infection model, bacterial lysates prepared by alkaline-hydrolysis had a 10 times higher immunostimulating effect than lysates prepared by acid-hydrolysis or heating . Bacterial lysates stimulated spontaneous cytotoxicity of natural mouse peritoneal killer cells after intraperitoneal application . Whole bacterial lysates had a higher NK-activity as their corresponding purified lipopolysaccharide portion.

Hum Genet, 1990 Sep, 85(4), 430 - 1
The delta F508 mutation in cystic fibrosis patients of southern Italy; Sebastio G et al.; Fifty one independent cystic fibrosis (CF) families originating from a restricted area of Southern Italy (Campania) have been analyzed for KM19 and XV2c haplotypes and the delta F508 mutation: 54% of the total CF chromosomes show the delta F508 mutation . No significative correlations were obtained when clinical score, radiological score, Pseudomonas colonization, or clinical symptoms at presentation were matched with the presence or absence of the delta F508 mutation.

J Bacteriol, 1990 Sep, 172(9), 4836 - 43
A cloned avirulence gene from Pseudomonas solanacearum determines incompatibility on Nicotiana tabacum at the host species level; Carney BF et al.; A locus in Pseudomonas solanacearum AW1 responsible for the hypersensitive response (HR) on tobacco was cloned by complementation in the tobacco-pathogenic strain P . solanacearum NC252 . The NC252 HR+ transconjugants lost pathogenicity on tobacco, indicating that the cloned locus could restrict the host range of NC252 . Restriction enzyme mapping, transposon mutagenesis, and subcloning showed that, at most, 2.0 kilobases of the cloned DNA was required for NC252 transconjugants to elicit HR on tobacco . Site-directed insertional mutagenesis of the wild-type locus in strain AW1 to create AW1-31 eliminated HR activity on tobacco . However, AW1-31 retained pathogenicity on tomato and eggplant, confirming that this locus contains an avirulence gene, designated avrA . In contrast to the wild type, AW1-31 multiplied to almost the same extent as NC252 after infiltration into tobacco leaves . Nevertheless, AW1-31 did not wilt tobacco when stem inoculated, suggesting that additional factors condition host range . AW1 was HR+ on 27 N . tabacum cultivars, whereas AW1-31 was always HR-, strongly suggesting that avrA is specific at the host species level.

Pediatr Med Chir, 1990 Sep-Oct, 12(5), 531 - 4
{Ciprofloxacin: an alternative oral treatment in respiratory Pseudomonas infection in cystic fibrosis}; Magni A et al.; 20 CF patients, aged from 16.5 to 31.7 years, with chronic pulmonary infection due to Pseudomonas, were included in an open trial to study the efficacy of ciprofloxacin on respiratory exacerbation . Ciprofloxacin was given orally at the dose of 1500 mg/die for ten days . 16 patients concluded the entire treatment with clear clinical improvement, based on a score including 11 parameters . There has also been a significant improvement in the pulmonary function tests, and a tendency of Rx score to decrease . 4 patients interrupted the treatment on the fifth day because of clinical inefficacy . There was no increase of Pseudomonas resistance to ciprofloxacin at the end of the treatment; 30 days after no strain of pseudomonas was found resistant . We observed side-effects in 5 patients, but in no case it was necessary to discontinue the treatment . Ciprofloxacin may be considered as a good alternative to the more established antibiotic strategy in the treatment of Pseudomonas lung exacerbations in CF.

Mikrobiol Zh, 1990 Sep-Oct, 52(5), 17 - 23
{The induction of tumor necrosis factor and interleukin-1 under exposure to the glycopolymers isolated from Pseudomonas solanacearum and Clavibacter michiganense}; Varbanets LD et al.; Lipopolysaccharide of Pseudomonas solanacearum and acid polysaccharides of Clavibacter michiganense are effective inductors of formation of the factor of tumour necrosis (TNF) and interleukin-1 (IL-1) by peritoneal macrophages of mice and their activity exceeds that of lipopolysaccharide Escherichia coli 055:B5 ("Sigma") . O-specific polysaccharide and lipid A are responsible for the capacity of liposaccharide molecule of P . solanacearum to induce TNF and IL-1.

Biochem Biophys Res Commun, 1990 Aug 31, 171(1), 1 - 6
TGF alpha-anti-Tac(Fv)-PE40: a bifunctional toxin cytotoxic for cells with EGF or IL2 receptors; Batra JK et al.; Conventional immunotoxins and chimeric toxins made in bacteria are directed to only one receptor or antigen on target cells . In this report we describe the construction of a chimeric molecule TGF alpha-anti Tac(Fv)-PE40 which is composed of human transforming growth factor type alpha attached to anti-Tac(Fv) which is in turn attached to PE40, a form of pseudomonas exotoxin, devoid of its cell recognition domain . TGF alpha-anti-Tac(Fv)-PE40 is a bifunctional toxin that is produced in E . coli and is active on cells bearing either IL2 or EGF receptors.

J Biol Chem, 1990 Aug 25, 265(24), 14675 - 83
Specificity and inhibition of proteases from human immunodeficiency viruses 1 and 2; Tomasselli AG et al.; Highly purified, recombinant preparations of the virally encoded proteases from human immunodeficiency viruses (HIV) 1 and 2 have been compared relative to 1) their specificities toward non-viral protein and synthetic peptide substrates, and 2) their inhibition by several P1-P1' pseudodipeptidyl-modified substrate analogs . Hydrolysis of the Leu-Leu and Leu-Ala bonds in the Pseudomonas exotoxin derivative, Lys-PE40, is qualitatively the same for HIV-2 protease as published earlier for the HIV-1 enzyme (Tomasselli, A . G., Hui, J . O., Sawyer, T . K., Staples, D . J., FitzGerald, D . J., Chaudhary, V . K., Pastan, I., and Heinrikson, R . L . (1990) J . Biol . Chem . 265, 408-413) . However, the rates of cleavage at these two sites are reversed for the HIV-2 protease which prefers the Leu-Ala bond . The kinetics of hydrolysis of this protein substrate by both enzymes are mirrored by those obtained from cleavage of model peptides . Hydrolysis by the two proteases of other synthetic peptides modeled after processing sites in HIV-1 and HIV-2 gag polyproteins and selected analogs thereof demonstrated differences, as well as similarities, in selectivity . For example, while the two proteases were nearly identical in their rates of cleavage of the Tyr-Pro bond in the HIV-1 gag fragment, Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val, the HIV-1 protease showed a 64-fold enhancement over the HIV-2 enzyme in hydrolysis of a Tyr-Val bond in the same template . Accordingly, the HIV-2 protease appears to have a different specificity than the HIV-1 enzyme; it is better able to hydrolyze substrates with small amino acids in P1 and P1', but is variable in its rate of hydrolysis of peptides with bulky substituents in these positions . In addition to these comparisons of the two proteases with respect to substrate specificity, we present inhibitor structure-activity data for the HIV-2 protease . Relative to P1-P1' statine or Phe psi {CH2N}Pro-modified pseudopeptidyl inhibitors, compounds having Xaa psi{CH(OH)CH2}Yaa inserts were found to show significantly higher affinities to both enzymes, generally binding from 10 to 100 times stronger to HIV-1 protease than to the HIV-2 enzyme . Molecular modeling comparisons based upon the sequence homology of the two enzymes and x-ray crystal structures of HIV-1 protease suggest that most of the nonconservative amino acid replacements occur in regions well outside the catalytic cleft, while only subtle structural differences exist within the active site.(ABSTRACT TRUNCATED AT 400 WORDS)

Arch Dis Child, 1990 Aug, 65(8), 874 - 7
Pseudomonas cepacia: a new pathogen in patients with cystic fibrosis referred to a large centre in the United Kingdom; Simmonds EJ et al.; Pseudomonas cepacia infection has become increasingly common among patients with cystic fibrosis in North America . In a large cystic fibrosis centre in the United Kingdom 11 cases have been identified during the last six years, with a maximum prevalence of 7% in 1988 . Three patients have died, two of whom deteriorated rapidly shortly after acquisition of the organism despite intensive treatment with appropriate antibiotics . Analysis of possible causes of the increase in P cepacia infection suggested that neither patient to patient transmission nor the use of nebulised antibiotics was associated with an increased risk of infection.

J Allergy Clin Immunol, 1990 Aug, 86(2), 231 - 8
Subclass distribution of IgG and IgA antibody response to Pseudomonas pseudoalcaligenes in humans exposed to infected metal-working fluid; Mattsby-Baltzer I et al.; The subclass distribution of the IgG and IgA antibody response in serum was studied in humans exposed to aerosolized metal-working fluid containing Pseudomonas pseudoalcaligenes . This species was consistently found in concentrations of 10(8) bacteria per milliliter of metal-working fluid during 1 year of observation . No increased frequency of respiratory infections or discomfort was related to the exposure to the infected fluid . The antibody response to the bacterium consisted predominantly of IgG1 and IgG2 antibodies . IgG2 antibodies dominated the antibody response to the lipopolysaccharide of the bacterium . IgA1 and IgA2 antibodies were also found . Smokers had significantly reduced antibody levels of all subclasses compared with nonsmokers . The antibody levels in smokers did not differ from levels of the unexposed control group . Analyses of the total serum immunoglobulin concentrations with respect to subclasses revealed that the total IgG2 levels were also significantly reduced in smokers . In nonsmokers, the age of the individuals influenced the antibody levels of the IgG1, IgG2, IgA1, and IgA2 subclasses, the levels decreasing with increasing age . For smokers, the correlation between age and antibody levels was only obvious for IgG2 antibodies . Decreased IgG2 antibody levels in the smokers were also accompanied by decreased FEV1 values (p less than 0.01) . Subclass analysis of the antibody response to P . pseudoalcaligenes demonstrated that the subclass pattern for the whole bacterium differed from the pattern of the major cell wall component, the lipopolysaccharide . The significance of qualitative and quantitative differences in the subclass antibody response is discussed.

Epidemiol Infect, 1990 Aug, 105(1), 127 - 37
Investigation of a pseudo-outbreak of 'Pseudomonas thomasii' in a special-care baby unit by numerical analysis of SDS-PAGE protein patterns; Costas M et al.; Forty-two cultures of pseudomonas comprising 28 clinical isolates from a pseudo-outbreak on a Special-Care Baby Unit and 14 reference strains, including 9 type strains, of various Pseudomonas species, were characterized by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins . The protein patterns were highly reproducible and were used as the basis for a numerical analysis which divided the strains into 9 phenons . Two of the 28 clinical isolates were identified by biochemical tests as P . pickettii and their identification was confirmed by SDS-PAGE as they fell in the same phenon as the type strain of the species . The remaining 26 isolates, which could not be identified on phenotypic tests, fell in the same phenon as three reference strains of 'P . thomasii' . The protein patterns provided the first clear evidence that P . pickettii and 'P . thomasii' were separate taxa and that the 'outbreak' was polymicrobial in origin, in line with the probable aqueous source of contamination . We conclude that high-resolution SDS-PAGE of proteins provides an effective method of identifying and differentiating pseudomonads, especially where this cannot be done adequately using conventional biochemical tests.

Proc Natl Acad Sci U S A, 1990 Aug, 87(15), 5797 - 801
Expression and fine structure of the gene encoding N epsilon-(indole-3-acetyl)-L-lysine synthetase from Pseudomonas savastanoi; Roberto FF et al.; The gene encoding N epsilon-(indole-3-acetyl)-L-lysine synthetase, iaaL, from Pseudomonas savastanoi was localized within a 4.25-kilobase EcoRI fragment derived from pIAA1 of oleander strain EW 2009 . Two open reading frames of 606 and 1188 nucleotides were identified upon sequencing, which directed the in vitro synthesis of Mr 21,000 and Mr 44,000 proteins . Expression of an open reading frame-2 subclone, pMON686, in Escherichia coli indicates that (indole-3-acetyl)-L-lysine synthetase is encoded solely by open reading frame-2 . Hydrophobicity plots of the deduced open reading frame-1 protein suggest that it may be a membrane-bound protein, whereas the predicted iaaL gene product possesses considerable hydrophilic character, consistent with the demonstration of (indole-3-acetyl)-L-lysine synthetase activity in cell-free aqueous extracts . No nucleotide or protein homologies were found between iaaL and any sequences contained within the GenBank or National Biomedical Research Foundation data bases (April 13, 1989).

J Bacteriol, 1990 Aug, 172(8), 4456 - 63
Formate dehydrogenase from the methane oxidizer Methylosinus trichosporium OB3b; Yoch DC et al.; Formate dehydrogenase (NAD+ dependent) was isolated from the obligate methanotroph Methylosinus trichosporium OB3b . When the enzyme was isolated anaerobically, two forms of the enzyme were seen on native polyacrylamide gels, DE-52 cellulose and Sephacryl S-300 columns; they were approximately 315,000 and 155,000 daltons . The enzyme showed two subunits on sodium dodecyl sulfate-polyacrylamide gels . The Mr of the alpha-subunit was 53,800 +/- 2,800, and that of the beta-subunit was 102,600 +/- 3,900 . The enzyme (Mr 315,000) was composed of these subunits in an apparent alpha 2 beta 2 arrangement . Nonheme iron was present at a concentration ranging from 11 to 18 g-atoms per mol of enzyme (Mr 315,000) . Similar levels of acid-labile sulfide were detected . No other metals were found in stoichiometric amounts . When the enzyme was isolated aerobically, there was no cofactor requirement for NAD reduction; however, when isolated anaerobically, activity was 80 to 90% dependent on the addition of flavin mononucleotide (FMN) to the reaction mixture . Furthermore, the addition of formate to an active, anoxic solution of formate dehydrogenase rapidly inactivated it in the absence of an electron acceptor; this activity could be reconstituted approximately 85% by 50 nM FMN . Flavin adenine dinucleotide could not replace FMN in reconstituting enzyme activity . The Kms of formate dehydrogenase for formate, NAD, and FMN were 146, 200, and 0.02 microM, respectively . "Pseudomonas oxalaticus" formate dehydrogenase, which has physical characteristics nearly identical to those of the M . trichosporium enzyme, was also shown to be inactivated under anoxic conditions by formate and reactivated by FMN . The evolutionary significance of this similarity is discussed.

Singapore Med J, 1990 Aug, 31(4), 335 - 7
Melioidosis: epidemiology and antibiogram of cases in Singapore; Tan AL et al.; There has been an increased incidence of melioidosis in Singapore . The disease affects mainly males, older patients and a disproportionately higher number of Indians and Malays . Possible predisposing illness include diabetes mellitus . Most patients are bacteraemic . Mortality rate is 72% for bacteraemic patients, as compared to 32% for non-bacteraemic patients . Local strains of Pseudomonas pseudomallei have been consistently sensitive to ceftazidime, chloramphenicol and piperacillin, and nearly always sensitive to tetracycline.

J Biochem (Tokyo), 1990 Aug, 108(2), 334 - 40
Activation of Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase by inorganic phosphate; Maruyama K; Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase {4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17}, one of the metal ion-requiring aldolases, is markedly activated by Pi . The activation is reversible and can be observed in every step of enzyme purification . The extent of activation is almost independent of the metal ion used, but varies with each substrate . The cleavage of l-4-carboxy-4-hydroxy-2-oxoadipate, a physiological substrate of the enzyme, is most strongly activated: Pi gives a hyperbolic activation curve with an activation constant of 0.36 mM and a maximum activation of about 65-fold . Arsenate, phosphorous acid, bicarbonate, acetyl phosphate, thiamine diphosphate, ADP, PPi, and ATP are also effective to various extents . These anions appear to be effective in the free form but not in the metal ion-complex . Many organic and inorganic anions are ineffective . Pi causes parallel increases in Vmax and in Km for substrate or metal ion with a concomitant shift of the optimum pH toward the alkaline side, and the enhancement of activity is closely correlated with the shift of optimum pH . Pi induces no gross change of molecular form of the enzyme protein as evaluated from gel filtration, PAGE, UV, fluorescence, and CD spectral data . Based on these findings, the mechanism and the physiological meaning of the observed activation are discussed.

J Biochem (Tokyo), 1990 Aug, 108(2), 327 - 33
Purification and properties of 4-hydroxy-4-methyl-2-oxoglutarate aldolase from Pseudomonas ochraceae grown on phthalate; Maruyama K; 4-Hydroxy-4-methyl-2-oxoglutarate aldolase {4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17} has been purified to homogeneity (about 770-fold purification, yield 11.4%) from Pseudomonas ochraceae grown on phthalate . The enzyme has a molecular weight of 160,000 (gel filtration on Bio-Gel A-1.5m), a subunit molecular weight of 26,000 (SDS-PAGE) and an isoelectric point of 5.0 (isoelectric focusing) . The enzyme requires divalent metal ions such as Mg2+, Mn2+, Co2+, Zn2+, and Cd2+ for activity . The enzyme actively cleaves 4-carboxy-4-hydroxy-2-oxoadipate, a physiological substrate of the enzyme, to give pyruvate and oxaloacetate, but shows much lower affinity for 4-hydroxy-4-methyl-2-oxoglutarate . 4-Hydroxy-2-oxoglutarate is cleaved at a low rate to pyruvate and glyoxylate . The l-isomers of the substrates are preferentially cleaved rather than the d-isomers as determined polarimetrically . The enzyme reactions are reversible: the equilibrium constants (pH 8.0, 25 C) for the HMG and HG cleavage reactions are about 0.07 and 0.03 M, respectively, whereas no equilibrium is observed with CHA due to oxaloacetate beta-decarboxylase activity associated with the enzyme . The enzyme activity is hardly affected by thiols and thiol reagents . The non-enzymatic cleavage reaction caused by various metal ions has also been studied to examine the mechanistic similarity to the enzymatic reaction.

FEMS Microbiol Lett, 1990 Aug, 58(3), 249 - 54
Cloning and expression of the carbon monoxide dehydrogenase genes from Pseudomonas thermocarboxydovorans strain C2; Black GW et al.; Carbon monoxide dehydrogenase (CODH) from Pseudomonas thermocarboxydovorans strain C2 is composed of three non-identical subunits . A gene library of C2 DNA in lambda vector L47.1 was generated and screened using anti-CODH serum . Western blotting experiments revealed a protein which co-migrated with and had the same immunological reaction as the large subunit of CODH in some of the clones isolated from the library . The coding region was pinpointed to a 4 kb fragment which was subcloned into plasmid . Western blotting experiments showed that all three subunits of CODH were coded for by the subclone . However, no CODH activity was detected.

J Clin Microbiol, 1990 Aug, 28(8), 1874 - 5
Evaluation of a modified complement fixation test and an indirect hemagglutination test for the serodiagnosis of melioidosis in pigs; Thomas AD et al.; A complement fixation test modified by the addition of porcine serum and an indirect hemagglutination test were used to detect antibodies to Pseudomonas pseudomallei in pigs . These tests together with cultural examinations were carried out with 250 pigs . The sensitivity and specificity values were 79.3 and 99.5% and 82.8 and 93.2% for the modified complement fixation and hemagglutination tests, respectively . When results from the combination of both tests were considered, the values were 86.2 and 92.8%, respectively.

Cryobiology, 1990 Aug, 27(4), 416 - 22
Clustering of ice nucleation protein correlates with ice nucleation activity; Mueller GM et al.; Antibodies raised against a synthetic peptide specifically detect ice nucleation proteins from Pseudomonas species in Western blots . In immunofluorescent staining of whole bacteria, the antibodies reveal the protein in clusters, as indicated by patches of intense fluorescence in Escherichia coli cells heterologously expressing Pseudomonas ice nucleation genes . The abundance, size, and brightness of the clusters vary considerably from cell to cell . Their varying sizes may explain the variability in activity of bacterial ice nuclei . Growth at lower temperatures produces more ice nuclei, and gives brighter and more frequent patches, than growth at 37 degrees C . The observed clustering may thus reflect formation of functional ice nucleation sites in vivo . The presence of ice nucleation protein in clusters is also correlated with alterations in cell morphology.

J Trauma, 1990 Aug, 30(8), 1000 - 5; discussion 1005-6
Fibrin glue-antibiotic suspension in the prevention of prosthetic graft infection; Ney AL et al.; The following study was done to assess whether fibrin glue-antibiotic suspension (FGAS) can prevent infection of a PTFE vascular graft in a contaminated wound . METHODS: FGAS was made by combining cryoprecipitate with a mixture of bovine thrombin, aminocaproic acid, and tobramycin (5 mg/cc thrombus) . Antibiotic activity was documented by in vitro kinetics which revealed initial elutions to be greater than 8,000 mu gm/cc and elutions at 4 days to be greater than 2 mcg/cc . Twelve dogs had a 1-cm section of infrarenal aorta replaced with a PTFE graft that had been bathed in a 2-cc solution of E . coli 3 x 10(8) CFU/ml and S . aureus 3 x 10(8) CFU/ml . Both organisms were sensitive to tobramycin and cefonicid . Dogs were divided into three groups of four . Group I had a contaminated PTFE graft placed and no further therapy . Group II had a contaminated PTFE graft placed and sealed with fibrin glue . Group III had a contaminated PTFE graft placed and sealed with FGAS . All three groups received daily IV cefonicid . RESULTS: Group I: Four of four dogs were reoperated on the fourth day for suspected sepsis and all four had pseudoaneurysms (one ruptured) . Three of four were culture positive for S . aureus and two of four positive for E . coli . Group II: Four of four died of anastomotic disruption by the third day . Four of four were culture positive for S . aureus and E . coli . Group III: All four dogs survived and were sacrificed on Day 17: all anastomoses were normal . Animal survival was significantly associated with the treatment given (p = 0.0025) . Three of four tissue cultures of the grafts were weakly positive for S . aureus and one of four for E . coli and Pseudomonas . Serum tobramycin levels were negligible at 12, 24, 72, and 96 hours . CONCLUSIONS: The data show that FGAS was associated with a reduction in vascular graft infection and pseudoaneurysm formation after exposure to a standardized bacterial inoculum . Whether complete eradication of all organisms can be achieved with higher doses of tobramycin is as yet undetermined.

Mol Gen Genet, 1990 Aug, 223(1), 33 - 9
Naphthalene degrading genes on plasmid NAH7 are on a defective transposon; Tsuda M et al.; A 37.5 kb region encompassing a set of the naphthalene degrading genes on the Pseudomonas plasmid NAH7 was found to be transposable only in the presence of the transposase encoded by the Tn1721 subgroup of the class II transposons . This newly identified mobile element, designated Tn4655, contained short (38 bp) terminal inverted repeats which shared extensive sequence homology with those of members of the Tn1721 subgroup . Tn4655 transposed by a two-step process involving formation of the cointegrate followed by its subsequent resolution . In contrast to the defect in the trans-acting factor for the first step, a functional system for the latter step was encoded within a 2.4 kb region in Tn4655 . Analysis of deletion and insertion mutants demonstrated that the 2.4 kb region contained the cis-acting (res) site and the gene for a trans-acting factor (resolvase); complementation analysis indicated that Tn4655 resolvase function was not interchangeable with those of other well-studied class II transposons, including the Tn1721 subgroup . Tn4655 had no DNA sequences that were hybridizable with the transposase or resolvase genes of the Tn1721 subgroup.

Biochim Biophys Acta, 1990 Aug 1, 1040(1), 12 - 8
Purification and properties of ampicillin acylase from Pseudomonas melanogenum; Kim DJ et al.; Ampicillin acylase, which is known to have a novel substrate spectrum, was purified to homogeneity from Pseudomonas melanogenum by the crude extract preparation and chromatography with S-Sepharose, hydroxyapatite, CM-cellulose C-52, and CM-Sepharose . The molecular weight of the native enzyme was calculated to be 146,000 by Protein PAK-300 sw HPLC chromatography . SDS-polyacrylamide gel electrophoresis revealed that the enzyme consisted of two identical subunits with a molecular weight of 72,000 . The enzyme was a glycoprotein containing 13% of total carbohydrate, and its isoelectric point was 7.2 . The enzyme catalyzed both synthesis and hydrolysis of ampicillin and hydrolysis of the ester bond of phenylglycinemethylester hydrochloride substrate . The substrate specificity showed that the enzyme required a free amino group on the alpha-carbon of the acyl group . Chemical modification by diethylpyrocarbonate or N-bromosuccinimide resulted in time-dependent inactivation of the enzyme, and other results suggest the participation of essential histidine residue(s) in the catalytic activity of ampicillin acylase . Substrates of the enzyme, 6-aminopenicillanic acid and ampicillin, exhibited protective effects against N-bromosuccinimide inactivation, suggesting that the modification occurred near or at the active site.

J Bacteriol, 1990 Aug, 172(8), 4728 - 31
Identification of a locus that regulates multiple functions in Pseudomonas solanacearum; Huang Y et al.; When Pseudomonas solanacearum K60 carries a multicopy plasmid containing cosmid clone pE6C (from the wild-type strain K60) or pBE6 (from the nonpathogenic strain B1), several phenotypic changes are observed, including the following: loss of virulence, reduced extracellular polysaccharide production, and increased polygalacturonase activity . Both cosmids contain a common 8-kilobase DNA region that is required for the phenotype shift . Saturation mutagenesis of pBE6 with Tn3-gus suggested that a single transcriptional unit of at least 1 kilobase is responsible for the phenotype shift . In maxicell assays, subclones containing this transcriptional unit expressed a single protein of about 25 kilodaltons.

South Med J, 1990 Aug, 83(8), 979 - 80
Imipenem resistance in a case of AIDS with relapsing Pseudomonas meningitis; Eng RH et al.; We describe an AIDS patient who had a recurrence of Pseudomonas meningitis to illustrate three points . First, the use of sulfamethoxazole-trimethoprim in AIDS patients for prophylaxis of Pneumocystis carinii pneumonia may cause the various body sites to be colonized with resistant species such as P aeruginosa . Second, Pseudomonas meningitis can recur in a patient with AIDS after a month of appropriate therapy . Finally, imipenem is a poor choice for Pseudomonas meningitis, even when alternative therapies appear much less attractive . High doses of imipenem should not be used for fear of seizures, and lower doses only produce resistant organisms in the CSF.

FEBS Lett, 1990 Jul 30, 268(1), 274 - 6
Multifrequency EPR evidence for a bimetallic center at the CuA site in cytochrome c oxidase; Kroneck PM et al.; Multifrequency electron paramagnetic resonance (EPR) spectra of the Cu(II) site in bovine heart cytochrome c oxidase (COX) and nitrous oxide reductase (N2OR) from Pseudomonas stutzeri confirm the existence of Cu-Cu interaction in both enzymes . C-band (4.5 GHz) proves to be a particularly good frequency complementing the spectra of COX and N2OR recorded at 2.4 and 3.5 GHz . Both the high and low field region of the EPR spectra show the presence of a well-resolved 7-line pattern consistent with the idea of a binuclear Cu center in COX and N2OR . Based on this assumption consistent g-values are calculated for gz and gx at four frequencies . No consistent g-values are obtained with the assumption of a 4-line pattern indicative for a mononuclear Cu site.

J Biol Chem, 1990 Jul 15, 265(20), 11628 - 32
Evidence that extracellular export of the endoglucanase encoded by egl of Pseudomonas solanacearum occurs by a two-step process involving a lipoprotein intermediate; Huang JZ et al.; Pseudomonas solanacearum is an important phytopathogen that produces a variety of extracellular enzymes . Previous reports suggested that one of these, a 43-kDa beta-1,4-endoglucanase (EGL), is initially synthesized with a 45-residue leader sequence that is removed during export . Experiments with globomycin presented here also suggest that the primary precursor of EGL (ppEGL) has a 45-residue leader sequence but that only the first 19 residues of the leader sequence are removed by signal peptidase II during initial export across the inner membrane . Further analysis suggested that the resultant 46-kDa intermediate precursor (pEGL) is a transient fatty acylated lipoprotein and is located on the periplasmic side of the inner membrane of P . solanacearum . Although Escherichia coli could synthesize ppEGL, modify it with palmitate, and remove the first 19 residues of the leader sequence during export across the inner membrane, only P . solanacearum could export pEGL across the outer membrane and remove the remaining 26 residues of the leader sequence producing the mature, extracellular EGL . The second step of the export process requires export machinery not present in E . coli . To our knowledge this represents the first example of a leader sequence with two distinct parts, one removed during export across the inner membrane and the other removed during export across the outer membrane.

Experientia, 1990 Jul 15, 46(7), 729 - 31
Expression of Pseudomonas phosphotriesterase activity in the fall armyworm confers resistance to insecticides; Dumas DP et al.; The gene encoding for the phosphotriesterase (opd) from Pseudomonas diminuta has been subcloned into a baculovirus expression system . Functional enzyme is produced when the recombinant baculovirus is used to infect either cultured Spodoptera frugiperda sf9 cells or the larval stage of the fall armyworm . The LD50 for paraoxon toxicity was found to increase 280-fold in the larvae after infection with the recombinant baculovirus and expression of the functional phosphotriesterase.

J Biol Chem, 1990 Jul 15, 265(20), 11783 - 7
The reaction of Pseudomonas nitrite reductase and nitrite . A stopped-flow and EPR study; Silvestrini MC et al.; The reaction between reduced Pseudomonas nitrite reductase and nitrite has been studied by stopped-flow and rapid-freezing EPR spectroscopy . The interpretation of the kinetics at pH 8.0 is consistent with the following reaction mechanism (where k1 and k3 much greater than k2) . {formula: see text} The bimolecular step (Step 1) is very fast, being lost in the dead time of a rapid mixing apparatus; the stoichiometry of the complex has been estimated to correspond to one NO2- molecule/heme d1 . The final species is the fully reduced enzyme with NO bound to heme d1; and at all concentrations of nitrite, there is no evidence for dissociation of NO or for further reduction of NO to N2O . Step 2 is assigned to an internal electron transfer from heme c to reduced NO-bound heme d1 occurring with a rate constant of 1 s-1; this rate is comparable to the rate of internal electron transfer previously determined when reducing the oxidized enzyme with azurin or cytochrome c551 . When heme d1 is NO-bound, the rate at which heme c can accept electrons from ascorbate is remarkably increased as compared to the oxidized enzyme, suggesting an increase in the redox potential of the latter heme.

Biochim Biophys Acta, 1990 Jul 6, 1039(3), 290 - 6
The solvent effects on the kinetics of bacterial formate dehydrogenase reaction; Demchenko AP et al.; The increase in concentration of organic cosolvents results in a 2-2.5-fold increase of the maximal reaction rate and a decrease of Michaelis constant for formate of NAD(+)-dependent formate dehydrogenase from methylotrophic bacteria Pseudomonas sp . 101 . These parameters, however, are not affected with the increase of ionic strength . For the logarithm of both Vmax and Km a linear function of the reciprocal of solvent dielectric permittivity was found . The decrease of Km is possibly due to the dielectric screening effect on the substrate binding energy . The increase in Vmax is explained by a model based on a solvent-dependent electrostatic image force, acting on the charges moved in the course of the catalytic step of the enzyme reaction.

J Heart Transplant, 1990 Jul-Aug, 9(4), 408 - 14
Treatment of cardiac allograft failure by use of an intraaortic axial flow pump; Frazier OH et al.; Since April 1988 we have used the Hemopump device, a new means of circulatory support, to successfully treat three orthotopic heart transplant recipients with biventricular failure refractory to conventional therapy . The Hemopump device is a 21F catheter-mounted, transvalvular, intraaortic axial flow pump . Power to the pump is percutaneously transmitted from an external electromechanical drive console by a flexible drive cable . We first used the pump in a 61-year-old man in whom severe steroid-resistant rejection developed 28 days after heart transplant, resulting in cardiogenic shock (cardiac index less than 2.0 L/min/m2) despite maximal inotropic support . In the second case a 49-year-old man with no evidence of pulmonary hypertension sustained cardiac arrest 2 hours after heart transplant, necessitating open chest massage and emergency cardiopulmonary bypass . The third patient was a 9-year-old boy in whom rejection developed 5 months after heart transplant, resulting in congestive heart failure that was unresponsive to maximal medical therapy . The device was implanted by way of the femoral artery approach in the first case, the ascending aorta in the second, and the distal abdominal aorta in the third . Duration of support was 46 hours, 65 hours, and 6 days, respectively . Increased blood flow provided by the pump ranged from 2 to 4 L/min . No device-related complications, such as hemolysis, infection, or thromboembolic events, occurred . All patients recovered normal heart function and were weaned from the device . The first patient is well after 12 months . The second patient died of metastatic lymphoma at 2 months, and the third died of Pseudomonas pneumonia after 2 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1990 Jul, 56(7), 2152 - 5
Fluoroacetate-metabolizing pseudomonad isolated from Dichapetalum cymosum; Meyer JJ et al.; A pseudomonad was isolated from the fluoroacetate-producing plant Dichapetalum cymosum (Hook) Engl . and identified as Pseudomonas cepacia . We established that this isolate was capable of growing in fluoroacetate-enriched solutions without any reduction in growth rate . Our isolate of P . cepacia was capable of defluorinating 2.69 mg of fluoroacetate per 10(9) cells per h . Fluoroacetate was degraded to CO2 at a rate of 23.53 ng/10(9) cells per h.

J Antibiot (Tokyo), 1990 Jul, 43(7), 783 - 7
Isolation of cepafungins I, II and III from Pseudomonas species; Shoji J et al.; New acylpeptide antibiotics named cepafungins I, II and III were isolated from the culture broth of a strain identified as Pseudomonas sp . These antibiotics are neutral substances, soluble in aqueous alcohols and dimethyl sulfoxide, and show UV maxima at 260.5 nm . The IR spectra indicated these to be peptides . Molecular formulas C28H46N4O6, C27H44N4O6 and C26H42N4O6 for cepafungins I, II and III were indicated by elemental analysis and SI-MS . Cepafungins exhibited inhibitory activity against yeast and fungi, and antitumor activity against P388 leukemia in mice.

Biochem J, 1990 Jul 1, 269(1), 25 - 9
Purification and characterization of a rat brain aldehyde dehydrogenase able to metabolize gamma-aminobutyraldehyde to gamma-aminobutyric acid; Abe T et al.; An enzyme which catalyses dehydrogenation of gamma-aminobutyraldehyde (ABAL) to gamma-aminobutyric acid (GABA) was purified to homogeneity from rat brain tissues by using DEAE-cellulose and affinity chromatography on 5'-AMP-Sepharose, phosphocellulose and Blue Agarose, followed by gel filtration . Such an enzyme was first purified from mammalian brain tissues, and was identified as an isoenzyme of aldehyde dehydrogenase . It has an Mr of 210,000 determined by polyacrylamide-gradient-gel electrophoresis, and appeared to be composed of subunits of Mr 50,000 . The close similarity of substrate specificity toward acetaldehyde, propionaldehyde and glycolaldehyde between the enzyme and other aldehyde dehydrogenases previously reported was observed . But substrate specificity of the enzyme toward ABAL was higher than those of aldehyde dehydrogenases from human liver (E1 and E2), and was lower than those of ABAL dehydrogenases from human liver (E3), Escherichia coli and Pseudomonas species . The Mr and relative amino acid composition of the enzyme are also similar to those of E1 and E2 . The existence of this enzyme in mammalian brain seems to be related to a glutamate decarboxylase-independent pathway (alternative pathway) for GABA synthesis from putrescine.

Ann Otol Rhinol Laryngol, 1990 Jul, 99(7 Pt 1), 543 - 6
Nasopharyngeal mass: a manifestation of inflammatory otologic disease; Metson R et al.; A nasopharyngeal mass with cranial neuropathies usually indicates an advanced neoplastic process . We present three patients with these findings and concurrent invasive Pseudomonas otitis in whom repeated nasopharyngeal biopsies were negative for tumor . All of the nasopharyngeal masses resolved following treatment of the otitis . Mechanisms of disease spread from the temporal bone to the nasopharynx are discussed . Clinicians may choose to modify diagnostic and therapeutic approaches to the nasopharyngeal mass in patients with concurrent invasive otologic disease.

J Bacteriol, 1990 Jul, 172(7), 3644 - 53
An operon containing the genes for cholesterol oxidase and a cytochrome P-450-like protein from a Streptomyces sp; Horii M et al.; The nucleotide sequence of the promoter region of the gene for cholesterol oxidase (choA) from Streptomyces sp . strain SA-COO was determined . We found an open reading frame (choP) that is located between a potential promoter sequence and the structural gene for the ChoA protein . Deletion analysis showed that the promoter region for choP is essential for expression of the choA gene . Mappings of S1 nuclease and primer extension of transcripts generated in vivo suggested that the synthesis of mRNA starts at a site 41 bases upstream from the ATG initiation codon of the choP gene . By Northern (RNA) blot analysis of the transcripts, we found a 2.9-kilobase transcript that is identical in size to the total sequence of the choP and choA genes . These results suggest that the two genes, choP and choA, are transcribed polycistronically under the control of the promoter that is upstream from the structural gene for choP . The choP gene encodes a protein of 381 amino acids with a calculated Mr of 41,668 . The nucleotide sequence of the choP gene has a high degree of similarity to the sequence of the genes for cytochrome P-450s from humans and Pseudomonas species . A region of homology with the cytochrome P-450s from various organisms was identified in the choP protein and may represent a region associated with a binding site for heme iron . Analysis of the CO difference spectrum of an extract of Streptomyces lividans cells that carry a plasmid which includes the choP gene revealed a unique peak, characteristic of cytochrome P-450, which is identical to that obtained with the parent strain.

Mol Gen Genet, 1990 Jul, 222(2-3), 461 - 6
Isolation and characterization of the gene from Pseudomonas syringae pv . phaseolicola encoding the phaseolotoxin-insensitive ornithine carbamoyltransferase; Mosqueda G et al.; The gene coding for the phaseolotoxin-insensitive ornithine carbamoyltransferase (OCTase) from Pseudomonas syringae pv . phaseolicola has been cloned and sequenced . The gene has a deduced coding capacity for a polypeptide with a calculated Mr of 36,520 daltons . Comparison of the amino acid sequence of the OCTase enzymes encoded by the P . aeruginosa argF and the Escherichia coli argI and argF genes with the deduced sequence of the newly identified gene shows that 79 amino acid residues are strictly conserved in all four polypeptides; among these 7 out of 9 residues are involved in enzyme function . Of three amino acid regions that have been implicated in substrate binding or catalysis, two are strictly conserved, and the third involved in carbamoylphosphate binding differs . This correlates well with published data showing that phaseolotoxin competes for the carbamoylphosphate binding site in the phaseolotoxin-sensitive OCTases . We propose that the gene be named argK.

Mikrobiol Zh, 1990 Jul-Aug, 52(4), 27 - 33
{The lipopolysaccharides of Pseudomonas solanacearum i Pseudomonas cichorii}; Varbanets LD et al.; Structure analysis by the methods of methylation, 1H- and 13C-n . m . r . spectroscopy has shown that O-specific polysaccharides of typical strains of Pseudomonas solanacearum (biovar I) and P . cichorii are identical by their structure and constructed of branched pentasaccharide repeating links which include three residues of rhamnose (one of them is in the branching node), one residue of beta-xylose (it occupies terminal position) and one reside of N-acetyl-beta-glucosamine . The other strain of P . solanacearum of biovar I and two strains belonging to biovars III and IV also produce structure-similar O-specific polysaccharides, constructed of linear tetrasaccharide repeating links which include three residues of alpha-L-rhamnose and one residue of N-acetyl-alpha-D-glucosamine.

Mikrobiologiia, 1990 Jul-Aug, 59(4), 616 - 23
{Physico-chemical properties of DNA from representatives of a population of Pseudomonas bacteriophages}; Romashko AM et al.; Twenty-four bacteriophages of Pseudomonas similar in terms of their morphology and structure were studied . The genomes of 23 bacteriophages from a population widespread in Belorussia and of one bacteriophage from the Krasnodar Region were shown to be double-helical DNAs with the content of GC base pairs from 46.8 to 66.7% . Four endonucleases (Eco R1, Hind III, Bam H1, and Eco RV) were used in the restriction analysis . Phage 8 which differed from the other phages in the area of its distribution was found to represent an individual group . The other phages were subdivided into eight related groups and had an identical restriction map within each group . The mean molecular masses of phage DNAs calculated by the method of summing the molecular masses of restricts ranged from 27.8 to 31.0 MDa.

Zhonghua Yan Ke Za Zhi, 1990 Jul, 26(4), 223 - 6
{Complications associated with soft hydrophilic contact lenses}; Shen BF; The authors report 89 cases of ocular complications associated with soft hydrophilic contact lenses, including corneal abrasions, corneal ulcers, punctate corneal epithelial erosions, aphakic corneal edema, and superior limbal keratoconjunctivitis . Soft contact lenses interfere with the integrity and function of the tear film, so that corneal metabolism is affected, predisposing the cornea to infections by Pseudomonas, Acanthamoebia, fungus and virus . The incidence of the complications is high; therefore, due attention should be devoted to their prevention and treatment.

Appl Environ Microbiol, 1990 Jul, 56(7), 2065 - 72
Starvation-specific formation of a peripheral exopolysaccharide by a marine Pseudomonas sp., strain S9; Wrangstadh M et al.; The marine bacterium Pseudomonas sp . strain S9 produces exopolysaccharides (EPS) during both growth and total energy source and nutrient starvation . Transmission electron microscopy of immunogold-labeled cells demonstrated that the EPS is closely associated with the cell surface during growth (integral EPS), while both the integral form and a loosely associated extracellular (peripheral) form were observed during starvation . Formation and release of the latter rendered the starvation medium viscous . In addition, after 3 h of starvation in static conditions, less than 5% of the cells were motile, compared with 100% at the onset of starvation and approximately 80% subsequent to release of the peripheral EPS at 27 h of starvation . Inhibition of protein synthesis with chloramphenicol added before 3 h of starvation caused no increase in viscosity . However, addition of chloramphenicol at 3 h did not prevent the subsequent increase in viscosity displayed by S9 cells . The amount of integral EPS increased for both nontreated and chloramphenicol-treated S9 cells during the first hour of starvation, with a subsequent equal decrease . The chloramphenicol-treated cells, as well as cells of a transposon-generated mutant strain deficient in peripheral EPS formation, remained adhesive to a hydrophobic inanimate surface during the initial 5 h of starvation, whereas nontreated wild-type cells had progressively decreased adhesion capacity . During the initial 5 h of starvation, most of the nontreated cells but only a small fraction of the chloramphenicol-treated and mutant cells detached from the hydrophobic substratum.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1990 Jul, 172(7), 3879 - 87
DNA sequence analysis of pglA and mechanism of export of its polygalacturonase product from Pseudomonas solanacearum; Huang JH et al.; The pglA gene encodes a 52-kilodalton extracellular polygalacturonase (PGA) which is associated with the phytopathogenic virulence of Pseudomonas solanacearum . The nucleotide sequence of pglA and the putative amino acid sequence of the PGA protein were determined . A computer search identified a 150-residue region of PGA which was similar (41%) to the amino acid sequence of a region of the PG-2A polygalacturonase from tomato . Comparison of the amino terminus of the pglA open reading frame with the actual amino-terminal sequence of purified extracellular PGA suggested that pglA is initially translated as a higher-molecular-mass precursor with a 21-residue amino-terminal signal sequence . Localization of various pglA-phoA fusion proteins in Escherichia coli and P . solanacearum indicated that the 21-residue leader sequence directs the export of PhoA only as far as the periplasm of both bacteria . Deletion of the last 13 residues of PGA eliminated its catalytic activity, as well as its ability to be exported outside of the P . solanacearum cell . Our results suggest that PGA excretion occurs in two steps . The first step involves a signal sequence cleavage mechanism similar to that used for periplasmic proteins and results in export of PGA across the inner membrane; the second step (transit of the outer membrane) occurs by an unknown mechanism requiring sequences from the mature PGA protein and biochemical factors absent from E . coli.

J Bacteriol, 1990 Jul, 172(7), 3946 - 51
Highly virulent strains of Pseudomonas solanacearum that are defective in extracellular-polysaccharide production; Xu PL et al.; Extracellular polysaccharide (EPS) has long been regarded as one of the most important factors involved in wilting of plants by Pseudomonas solanacearum . By means of transposon Tn5 mutagenesis, we have isolated a class of mutants that have an afluidal colony morphology but retain the ability to cause severe wilting and death of tobacco plants . One such mutant, KD700, was studied in detail . By marker exchange mutagenesis, the altered colony morphology was shown to be the result of a single Tn5 insertion in a 14.3-kilobase EcoRI fragment . This defect could be corrected by introducing a homologous clone from a cosmid library of the wild-type, parental strain K60 . The Tn5-containing fragment was introduced into other P . solanacearum wild-type strains by marker exchange, and these altered strains had the same afluidal phenotype as KD700 . N-Acetylgalactosamine (GalNac), the major constituent of EPS of all wild-type strains of P . solanacearum, was not detected by gas chromatography-mass spectrometry analysis of vascular fluids from wilting plants infected by KD700 . In contrast, GalNac was readily detected in similar fluids of plants infected by K60 . Polysaccharides extracted from culture filtrates of KD700 contained approximately one-fifth of the GalNac present in polysaccharides from K60 . No differences in growth rates in culture or in planta between the mutant and the parental strains were observed . Since strains that are deficient in EPS production can remain highly virulent to tobacco, we conclude that EPS, or at least its GalNac-containing component, may not be required for disease development by P . solanacearum.

J Virol, 1990 Jul, 64(7), 3157 - 61
Proteases from human immunodeficiency virus and avian myeloblastosis virus show distinct specificities in hydrolysis of multidomain protein substrates; Tomasselli AG et al.; The virally encoded proteases from human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) have been compared relative to their ability to hydrolyze a variant of the three-domain Pseudomonas exotoxin, PE66 . This exotoxin derivative, missing domain I and referred to as LysPE40, is made up of a 13-kilodalton NH2-terminal translocation domain II connected by a segment of 40 amino acids to enzyme domain III of the toxin, a 23-kilodalton ADP-ribosyltransferase . HIV protease hydrolyzes two peptide bonds in LysPE40, a Leu-Leu bond in the interdomain region and a Leu-Ala bond in a nonstructured region three residues in from the NH2-terminus . Neither of these sites is cleaved by the AMV enzyme; hydrolysis occurs, instead, at an Asp-Val bond in another part of the interdomain segment and at a Leu-Thr bond in the NH2-terminal region of domain II . Synthetic peptides corresponding to these cleavage sites are hydrolyzed by the individual proteases with the same specificity displayed toward the protein substrate . Peptide substrates for one protease are neither substrates nor competitive inhibitors for the other . A potent inhibitor of HIV type 1 protease was more than 3 orders of magnitude less active toward the AMV enzyme . These results suggest that although the crystallographic models of Rous sarcoma virus protease (an enzyme nearly identical to the AMV enzyme) and HIV type 1 protease show a high degree of similarity, there exist structural differences between these retroviral proteases that are clearly reflected by their kinetic properties.

Appl Environ Microbiol, 1990 Jul, 56(7), 2056 - 64
Identification of an additional ferric-siderophore uptake gene clustered with receptor, biosynthesis, and fur-like regulatory genes in fluorescent Pseudomonas sp . strain M114; O'Sullivan DJ et al.; Five cosmid clones with insert sizes averaging 22.6 kilobases (kb) were isolated after complementation of 22 Tn5-induced Sid- mutants of Pseudomonas sp . strain M114 . One of these plasmids (pMS639) was also shown to encode ferric-siderophore receptor and dissociation functions . The receptor gene was located on this plasmid since introduction of the plasmid into three wild-type fluorescent pseudomonads enabled them to utilize the ferric-siderophore from strain M114 . The presence of an extra iron-regulated protein in the outer membrane profile of one of these strains was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . A ferric-siderophore dissociation gene was attributed to pMS639 since it complemented the ferric-siderophore uptake mutation in strain M114FR2 . This mutant was not defective in the outer membrane receptor for ferric-siderophore but apparently accumulated ferric-siderophore internally . Since ferric-citrate alleviated the iron stress of the mutant, there was no defect in iron metabolism subsequent to release of iron from the ferric-siderophore complex . Consequently, this mutant was defective in ferric-siderophore dissociation . A fur-like regulatory gene also present on pMS639 was subcloned to a 7.0-kb BglII insert of pCUP5 and was located approximately 7.3 kb from the receptor region . These results established that the 27.2-kb insert of pMS639 encoded at least two siderophore biosynthesis genes, ferric-siderophore receptor and dissociation genes, and a fur-like regulatory gene from the biocontrol fluorescent Pseudomonas sp . strain M114.

Plant Mol Biol, 1990 Jul, 15(1), 145 - 54
Transcriptional activation of 2 classes of genes during the hypersensitive reaction of tobacco leaves infiltrated with an incompatible isolate of the phytopathogenic bacterium Pseudomonas solanacearum; Marco YJ et al.; Fourteen cDNA clones whose corresponding mRNAs accumulate during the hypersensitive reaction (HR) of tobacco leaves infiltrated with an incompatible strain of the bacterial pathogen Pseudomonas solanacearum have been subdivided by sequence homologies into 6 families . Studies on the accumulation of the mRNAs encoded by these genes in compatible and incompatible plant-bacterial interactions have been carried out and indicate that the 6 cDNA clones can be subdivided into 2 groups . In one group corresponding to 3 cDNA clones, the maximal level of mRNA accumulation is similar in both types of interaction, whereas in the other group, maximal mRNA accumulation in leaves undergoing an HR is 3- to 7-fold higher than in leaves infiltrated with the compatible strain . Within each group, the timing and kinetics of accumulation of the corresponding mRNAs differ for each individual cDNA clone . Run-on experiments indicate that transcriptional activation of these genes plays a major role in the control of their expression . Genomic hybridizations have been performed and indicate that the mRNAs corresponding to the cDNA clones are encoded by multigene families (6 to 20 genes).

Rev Argent Microbiol, 1990 Jul-Sep, 22(3), 155 - 8
{Loquat canker: a new disease for Argentina}; Alippi AM et al.; A stem canker disease caused by Pseudomonas syringae pv . eriobotryae (Takimoto) Young, Dye y Wilkie on loquat (Eriobotrya Japonica {Thumb} Lindl) was recorded for the first time in Argentina . Symptoms of the disease appeared as dry stem cankers which in advanced stages surrounded the stems . Similar cankers were noticeable on leaves midribs . Seven bacterial strains were isolated from diseased loquats and their identification was based on disease symptoms, pathogenicity and cultural and biochemical characteristics . All strains were levan positive and gave a hypersensitive reaction on tobacco leaves . Neither arginine dehydrolase nor oxidase was detected in any of the strains which produced a diffusible green pigment on King B which fluoresced under UV light and a distinct diffusible brown pigment on King B, SPA and Tween 80 media within 5-7 days of incubation . Lipolysis of Tween 80 was also recorded . The symptoms observed in the field and obtained by experimental inoculations were similar to those induced by Pseudomonas syringae pv . eriobotryae in the original description of the disease.

Bioconjug Chem, 1990 Jul-Aug, 1(4), 264 - 8
Antitumor activity of a thioether-linked immunotoxin: OVB3-PE; FitzGerald D et al.; A thioether-linked immunotoxin was made between Pseudomonas exotoxin and the monoclonal antibody OVB3 . This conjugate, OVB3-PE, was cytotoxic for the human ovarium cancer cell line OVCAR-3 (ID of 2.5 x 10(-12) M) and it was therefore tested for antitumor activity in a nude mouse model of ovarian cancer . This model employs the injection of a lethal number of OVCAR-3 cells into the peritoneal cavity of nude mice . When 0.2-1 micrograms of OVB3-PE was injected intraperitoneally on three successive days beginning 3-5 days after OVCAR-3 cell implantation, the survival of the tumor-bearing mice was increased 2-4-fold compared to that of untreated control mice . Median survival times for control mice ranged from 44 to 50 days while survival times of 150 days or greater were seen in mice treated with OVB3-PE . When OVB3-PE administration was delayed until 2-4 weeks after tumor cell implantation, OVB3-PE treatment also showed antitumor activity, but the duration of survival was less than with the early treatments . OVB3-PE was also cytotoxic for MCF-7 breast carcinoma cells, HT-29 colon carcinoma cells, and A431 epidermoid carcinoma cells.

Trans R Soc Trop Med Hyg, 1990 Jul-Aug, 84(4), 585 - 7
The use of bone marrow culture for the diagnosis of melioidosis; Dance DA et al.; We have evaluated prospectively the contribution of bone marrow culture to the diagnosis of melioidosis . Bone marrow (BMC) and blood cultures (BC) were collected concurrently from 105 patients with suspected acute, severe melioidosis . 67 patients were subsequently proved to have the disease whilst other significant organisms were isolated from these specimens in 5 cases . Overall, 67.2% of BC and 64.2% of BMC from melioidosis patients grew Pseudomonas pseudomallei . Time to positivity did not differ significantly in paired BC and BMC specimens . These results do not support the routine use of BMC in the diagnosis of acute, severe melioidosis . In one patient with pulmonary melioidosis, however, blood cultures were repeatedly negative, whilst bone marrow grew P . pseudomallei, and this preceded the development of a distant focus of infection . This suggests that culture of bone-marrow may be of value in certain blood culture-negative patients with melioidosis.

J Hosp Infect, 1990 Jul, 16(1), 59 - 65
A pseudo-outbreak of Pseudomonas on a special care baby unit; Heard S et al.; Following isolation of a multi-antibiotic-resistant pseudomonad from a newborn infant on admission to the Special Care Baby Unit and further isolation of apparently the same organism from two additional infants, a full investigation was instigated in an attempt to discover the source of the organism . This revealed a further 13 infants apparently colonized with the same organism . Repeated screening of the infants with a commercial sterile swab/transport medium failed to isolate the organism . Examination of bottles of the in-house transport medium, which had been stored under a sink, produced further isolates of the same organism . Water splashed from the sink was suspected as the ultimate source of contamination . Biochemical characterization showed that P . pickettii and at least one other Pseudomonas species were involved . The epidemiological, clinical and economic implications of the 'outbreak' are discussed together with the ultimate financial implications for investigation of such incidents.

Mikrobiol Zh, 1990 Jul-Aug, 52(4), 71 - 4
{Glycopolymers of Clavibacter michiganense and Pseudomonas solanacearum--interferon inducers}; Varbanets LD et al.; A polysaccharide, being an active inductor of gamma-interferon, has been found among glycopolymers of Clavibacter michiganense . Lipopolysaccharide of Pseudomonas solanacearum is shown to display an interferonogenic activity which is analogous to that of the classical interferon inductor, lipopolysaccharide of Escherichia coli O55: B5 . Besides lipid A, core oligosaccharide also contributes much to the interferonogenic activity of P . solanacearum lipopolysaccharide.

Biochim Biophys Acta, 1990 Jun 21, 1049(2), 227 - 30
Nucleotide sequences of the meta-cleavage pathway enzymes 2-hydroxymuconic semialdehyde dehydrogenase and 2-hydroxymuconic semialdehyde hydrolase from Pseudomonas CF600; Nordlund I et al.; The nucleotide sequence of a 2493 base pair (bp) region, spanning the coding regions for the meta-cleavage pathway enzymes 2-hydroxymuconic semialdehyde dehydrogenase (HMSD) and 2-hydroxymuconic semialdehyde hydrolase (HMSH), was determined . The deduced protein sequence for HMSD is 486 amino acid residues long with an Mr of 51,682 . HMSD has homology with a number of aldehyde dehydrogenases from various eukaryotic sources . The deduced protein sequence for HMSH is 283 amino acids long with an Mr of 30,965 . The amino acid composition of this enzyme is similar to that of isofunctional enzymes from toluene and m-cresol catabolic pathways.

Med J Aust, 1990 Jun 18, 152(12), 652 - 5
An Australia-wide epidemic of Pseudomonas pickettii bacteraemia due to contaminated "sterile" water for injection; Roberts LA et al.; Nineteen cases of Pseudomonas pickettii bacteraemia and one case of Pseudomonas cepacia bacteraemia were identified in an Australia-wide outbreak of nosocomial sepsis associated with contaminated water for injection . The contamination was limited to one batch of commercially produced water for injection . Four different organisms were identified (three biotypes of P . pickettii and one of P . cepacia) . However, P . pickettii biotype 1 appeared to be relatively more virulent than the other biotypes as it was the only identified organism in blood cultures in nearly all cases of sepsis . The ampoules of "sterile" water were each contaminated with approximately 10(3) organisms per millilitre . The lack of an Australian central reporting system for bacteraemia delayed the recognition of this outbreak.

Rev Prat, 1990 Jun 11, 40(17), 1581 - 6
{Cystic fibrosis in adults}; Polu JM et al.; While fifty years ago 20 p . 100 of cystic fibrosis patients only reached the age of one year, more than 50 p . 100 of the patients now live more than twenty years . The clinical manifestations of cystic fibrosis are more diverse in adults than in children, so that the diagnosis might concern several specialties . In actual fact, only 3 to 7 p . 100 of cystic fibroses are diagnosed after thirteen to sixteen years, and in half the cases the symptoms had been present before the age of one year . In adults, the respiratory manifestations of cystic fibrosis are predominant, whereas the gastrointestinal manifestations tend to be blurred . Radiography of the chest shows interstitial lesions (opacities, cystic images, disorders of ventilation), principally located in the right side and the apex . The most common functional defect is an obstructive syndrome corresponding to a gradual involvement of the peripheral airways . A number of complications may develop, including recurrent Pseudomonas infection of the lung, pneumothorax, heart failure, malnutrition, liver cirrhosis, episodes of intestinal occlusion, etc . The longer life span of these patients raises the problems of diabetes with its vascular complications, infertility or pregnancy, social and professional insertion, and so forth . The prognosis of cystic fibrosis in adults depends on the date the diagnosis was made, on the therapeutic follow-up and on the creation of specialized centres . The control of Pseudomonas infections and the development of lung transplantation are the main advances to be expected.

J Clin Microbiol, 1990 Jun, 28(6), 1120 - 4
Pneumonia caused by a newly recognized pseudomonad in a child with chronic granulomatous disease; Trotter JA et al.; A pseudomonad was isolated from the pleural fluid and pulmonary decortication tissue of a 5-year-old child with chronic granulomatous disease . Although the isolate was phenotypically similar to Pseudomonas cepacia, its biochemical profile was more similar to that of Pseudomonas pickettii biovar 2 . Its slow growth rate, ability to hydrolyze urea rapidly, and lateral and polar flagellar pattern were suggestive of Oligella ureolytica (formerly CDC group IVe) . The cellular fatty acid composition was similar to that of P . cepacia and Pseudomonas gladioli, except for the presence of dodecanoic acid . Numerical analysis of the fatty acid data supported the interrelatedness of the isolate with other species of the pseudomallei group (rRNA homology group II) of Pseudomonas . The organism described in this report is an addition to the growing list of catalase-positive organisms which can potentially cause severe morbidity in patients with chronic granulomatous disease.

Proc Natl Acad Sci U S A, 1990 Jun, 87(12), 4697 - 701
Transforming growth factor alpha-Pseudomonas exotoxin fusion protein prolongs survival of nude mice bearing tumor xenografts; Heimbrook DC et al.; Transforming growth factor alpha (TGF alpha)-Pseudomonas exotoxin 40 (PE40) is a chimeric protein consisting of an N-terminal TGF alpha domain fused to a C-terminal 40-kDa segment of the Pseudomonas exotoxin A protein . TGF alpha-PE40 exhibits the receptor-binding activity of TGF alpha and the cell-killing activity of PE40 . These properties make TGF alpha-PE40 an effective cytotoxic agent for cells that possess epidermal growth factor receptors (EGFR) . However, the utility of this protein as an anticancer agent has been unclear because many normal tissues express EGFR and may be damaged by exposure to TGF alpha-PE40 . To address this issue, we injected nude mice with a lethal inoculum of either A431 or HT29 human tumor cells that possess EGFR or with Chinese hamster ovary (CHO) tumor cells that lack EGFR . Animals were treated with a derivative of TGF alpha-PE40 in which the cysteine residues are replaced by alanine, termed "TGF alpha-PE40 delta cys," or with saline once a day for 5 days . Mice bearing EGFR+ tumor cells lived significantly (P less than 0.001) longer when treated with TGF alpha-PE40 delta cys compared with saline-treated controls (median survival: A431 cells, 51.5 vs . 25.5 days; HT29 cells, 101 vs . 47.5 days) . TGF alpha-PE40 delta cys did not prolong the survival of mice bearing tumor cells that lack EGFR (median survival: CHO cells, 15.5 vs . 19.5 days) . The only toxicity to normal tissues was mild periportal hepatic necrosis . These studies indicate that a therapeutic window exists in vivo for the use of some growth factor-toxin fusion proteins as anticancer agents.

J Invest Dermatol, 1990 Jun, 94(6 Suppl), 158S - 163S
Purification of the IL-2 receptor (TAC) by ligand-affinity chromatography and utilization of the immobilized receptor for receptor-affinity chromatography (RAC) purification of IL-2, mutant IL-2, and IL-2 fusion proteins; Smart JE et al.; Recombinant technology has facilitated the production of two soluble forms of human p55 interleukin-2 receptor (IL-2R) in Chinese hamster ovary cells . We have developed a ligand-affinity method for the medium-scale purification of these two soluble forms of the IL-2R, based on the biochemical interactions between the matrix-bound ligand (interleukin-2) and its soluble receptor . The affinity-purified IL-2R is further purified by anion-exchange chromatography followed by gel filtration . This method has provided enough highly pure IL-2R for structure and function studies and for use in practical applications such as high-flux drug-screening assays . The purified IL-2R subsequently has been immobilized on silica gel and employed for the purification of recombinant IL-2 . Receptor-affinity-chromatography-purified IL-2 contains only a highly active monomeric form of the lymphokine, in contrast to immunoaffinity chromatography where several molecular forms of IL-2 with varying degrees of biologic activity are recovered . Receptor-affinity chromatography has been successfully applied to the purification of several mutant IL-2 as well as an IL-2-Pseudomonas exotoxin (IL2-PE40) fusion protein that is a 54.5-kDa chimeric protein in which the cell recognition domain is replaced by IL-2 . The IL-2-PE40 is a potential cytotoxic agent for cells bearing the IL-2 receptor.

Am J Surg, 1990 Jun, 159(6), 579 - 81
Continuous ambulatory peritoneal dialysis associated with peritonitis in older patients; Valente J et al.; Our recent experience with peritonitis in patients over the age of 55 years undergoing continuous ambulatory peritoneal dialysis between 1979 and 1989 is reviewed . Thirty-seven patients in this age group underwent Tenckhoff catheter insertion . Severe catheter-related peritonitis occurred at a rate of 1.41 episodes per patient per year . Overall, there were 61 episodes of peritonitis in 31 patients, with an overall mortality rate of 7% . When systemic signs of sepsis were present, this rate rose to 25% . All deaths were associated with fungal, pseudomonal, or polymicrobial infections . Management of these infections may require aggressive measures including repeated laparotomy for control of sepsis.

Int J Biol Macromol, 1990 Jun, 12(3), 195 - 200
Conformational transition and polyelectrolyte behaviour of a succinoglycan polysaccharide; Gravanis G et al.; We report the chemical characterization and the relationship between the physicochemical properties and conformational change of a succinoglycan polysaccharide produced by Pseudomonas sp, NCIB 11592 . The expected chemical structure is confirmed, with a ratio of D-glucose: D-galactose: pyruvate: succinate of 7:1:1:1 . The molecular weight of the native form is 4.2 x 10(6) but after a single heating cycle through the disordered state the molecular weight is reduced to 3.0 x 10(6) . The polymer has a polymolecularity index of 1.3 in both cases . The conformational change was studied by different methods which enabled us to define the exact nature of the ordered and disordered states . The conformational transition depends on the temperature, the ionic strength and the nature of the counterion . The polyelectrolyte behaviour is in favour of a single chain conformation with an intramolecular helix-coil transition . The enthalpy change during this transition is greater than that expected solely on the basis of the polyelectrolyte contribution . It may be associated with changes in solvation or a rearrangement of water molecules in close association with the polymer.

Bull Soc Ophtalmol Fr, 1990 Jun-Jul, 90(6-7), 645 - 7
{Pyocyanic corneal abscess}; Khaitrine L et al.; The authors report 3 different cases of pseudomonas abscess of the corneal layers: these occurred; one on a soft lens; the second one apparently without any reason; the last one 2 days after a corneal traumatism . In spite of rapidly administrated antibiotherapy, the abscess spread very rapidly and left a residual corneal macula.

FEMS Microbiol Lett, 1990 Jun 1, 57(3), 317 - 21
Dioxygenolytic cleavage of aryl ether bonds: 1,2-dihydro-1,2-dihydroxy-4-carboxybenzophenone as evidence for initial 1,2-dioxygenation in 3- and 4-carboxy biphenyl ether degradation; Engesser KH et al.; A bacterial strain, Pseudomonas sp . POB 310, was enriched with 4-carboxy biphenyl ether as sole source of carbon and energy . Resting cells of POB 310 co-oxidize a substrate analogue, 4-carboxybenzophenone, yielding 1,2-dihydro-1,2-dihydroxy-4-carboxy-benzophenone . The ether bond of 3- and 4-carboxy biphenyl ether is cleaved analogously by initial 1,2-dioxygenation, yielding a hemiacetal which is hydrolysed to protocatechuate and phenol . These intermediates are degraded via an ortho and meta pathway, respectively . Alternative 2,3- and 3,4-dioxygenation can be ruled out as triggering steps in carboxy biphenyl ether degradation.

J Clin Microbiol, 1990 Jun, 28(6), 1249 - 53
Enzyme-linked immunosorbent assay for immunoglobulin M specific antibody for the diagnosis of melioidosis; Kunakorn M et al.; Indirect hemagglutination (IHA) is commonly used for serodiagnosis of melioidosis . However, in endemic areas, high background titers in normal populations and occasional low titers in patients with septicemic melioidosis prompted a search for a more sensitive and more specific method of serodiagnosis . An indirect fluorescent-antibody test for immunoglobulin M (IgM) specific antibody to Pseudomonas pseudomallei was more sensitive and more specific, but fluorescence microscopes are rarely available in the endemic areas . An enzyme-linked immunosorbent assay (ELISA) for IgM antibody is an attractive alternative . An indirect ELISA for IgM antibody (IgM ELISA) and an IgM antibody capture ELISA for melioidosis were developed . Both tests, together with IHA, were evaluated for 153 serum specimens from blood donors and 16 serum specimens from 16 melioidosis patients . It was found that IHA, the IgM ELISA, and the IgM antibody capture ELISA had sensitivities of 88, 88, and 75%, respectively, with specificities of 97.4, 92.2, and 91.5%, respectively . When IHA was combined with IgM ELISA, a sensitivity of 100% and a specificity of 95.4% were obtained . The IgM ELISA and IHA should be used in combination for serodiagnosis of melioidosis.

J Antimicrob Chemother, 1990 Jun, 25(6), 959 - 63
Cefepime concentrations in bronchial mucosa and serum following a single 2 gram intravenous dose; Chadha D et al.; The concentrations of cefepime in bronchial mucosa were measured after intravenous administration of a single 2 g dose in 20 patients undergoing diagnostic fibreoptic bronchoscopy . These concentrations were compared with simultaneous serum concentrations . The mean bronchial mucosal concentration was 24.1 mg/kg (s.d . 17.8 mg/kg) and the mean serum concentration was 40.4 mg/l (s.d . 28.1 mg/l) . The mean percentage penetration was 59.8% (s.d . 12.5%) . We conclude that a twice daily dosing of cefepime would be adequate for most respiratory infections although an 8-hourly dose may be necessary in pseudomonal infections.

J Trauma, 1990 Jun, 30(6), 737 - 40
Antibiotic prophylaxis diminishes bacterial translocation but not mortality in experimental burn wound sepsis; Jones WG 2nd et al.; Pseudomonas (PSA) burn wound sepsis results in prolonged bacterial translocation (BT) of enteric organisms such as E . coli to the mesenteric lymph nodes (MLN) and organs in rats . Intestinal decontamination with oral antibiotics may improve mortality after burn injury, perhaps due to decreased BT . To determine the effect of oral antibiotic prophylaxis effective against E . coli but not PSA on BT and subsequent mortality in a model of PSA burn wound sepsis, rats were given a 30% scald burn and wound inoculation with 10(8) PSA followed by randomization to either ampicillin (50 mg/kg/d) or saline gavage . Cultures of MLN, organs, blood, and cecal contents were obtained on days 1, 4, and 7 after injury, with additional animals observed for 14-day mortality . Although oral antibiotic prophylaxis resulted in increased cecal colony counts, the incidence of BT was unchanged . The number of organisms present in both the MLN and organs, however, was significantly reduced with prophylaxis, indicating cecal overgrowth by non-translocating bacteria . Reduction of the number of translocating organisms did not result in improved mean survival time after injury, suggesting that mortality from PSA burn wound sepsis occurs independently of bacterial translocation.

FEMS Microbiol Lett, 1990 Jun 1, 57(3), 323 - 8
A catabolic plasmid involved in 4-methyl-o-phthalate and 4-hydroxy-iso-phthalate degradation in Pseudomonas cepacia; Saint CP et al.; Genes involved in 4-methyl-o-phthalate and 4-hydroxy-iso-phthalate catabolism reside on a 226-232 kbp catabolic plasmid termed MOP . This was confirmed by transformation and conjugation into an isogenic heat-cured (MOP-) derivative of the wild-type isolate, identified and termed Pseudomonas cepacia Pc701 . Transformation confirmed the presence of Tn1 in MOP derived from Pc704, a mutant deficient in 4-methyl-o-phthalate catabolism . pCS1, a recombinant plasmid bearing MOP DNA, complemented MOP::Tn1 restoring the ability of Pc704 to grow on 4-methyl-o-phthalate . DNA-DNA hybridization using pCS1 as probe confirmed that loss of 4-methyl-o-phthalate catabolism by Pc704 was the result of Tn1 insertion into a 2.1 kbp HindIII fragment of MOP.

Mol Cell Biol, 1990 Jun, 10(6), 2443 - 7
Cell-specific toxicity of a chimeric protein composed of interleukin-6 and Pseudomonas exotoxin (IL6-PE40) on tumor cells; Siegall CB et al.; IL6-PE40 is a chimeric toxin composed of human interleukin-6 (IL6) linked by a peptide bond to PE40, a form of Pseudomonas exotoxin (PE) devoid of its cell recognition domain . To identify cancer cell lines with high numbers of IL6 receptors and to assess the usefulness of IL6-PE40 as a possible anticancer agent, we evaluated the toxicity of IL6-PE40 on a variety of tumor cell lines and demonstrated that certain human myeloma and hepatoma cell lines were particularly sensitive . IL6 binding to selected hepatoma and myeloma cell lines were determined by using {125I}IL6 . IL6 receptor mRNA levels were measured by polymerase chain reactions . When comparisons were made among different hepatoma cell lines, the sensitivity to IL6-PE40 correlated with the number of IL6 receptors . However, the hepatoma line PLC/PRF/5, which contains 2,300 IL6 receptors, was more sensitive to IL6-PE40 (amount of protein required to inhibit protein synthesis by 50% was 5 ng/ml) than both the myeloma cell lines U266 and H929 (for both cell lines, the 50% inhibitory dose was 8 ng/ml), which contain 15,500 and 16,500 IL6 receptors, respectively . RNA analysis confirmed that the sensitivity of these cells to IL6-PE40 and the amount of IL6 receptor RNA detected did not correlate . These data suggest that factors in addition to the number of IL6-binding sites contribute to the sensitivity of cells to IL6-PE40.

Mol Cell Probes, 1990 Jun, 4(3), 175 - 87
Detection of Legionella with polymerase chain reaction and gene probe methods; Mahbubani MH et al.; Methods were developed for the detection of Legionella in environmental water sources, based upon the polymerase chain reaction (PCR) and gene probes . All species of Legionella, including all 15 serogroups of L . pneumophila tested, were detected by PCR amplification of a 104 bp DNA sequence that codes for a region of 5S rRNA followed by radiolabelled oligoprobe hybridization to an internal region of the amplified DNA . Strains of L . pneumophila (all serogroups) were specifically detected based upon amplification of a portion of the coding region of the macrophage infectivity potentiator (mip) gene . Pseudomonas spp . that exhibit antigenic cross-reactivity in serological detection methods did not produce positive signals in the PCR-gene probe method using Southern blot analyses . Single cell, single gene Legionella detection was achieved with the PCR-gene probe methods.

Gene, 1990 May 31, 90(1), 145 - 8
Construction of broad-host-range vectors for the selection of divergent promoters; Ronald SL et al.; A series of promoter-probe plasmid vectors has been constructed which allows for the selection of DNA sequences containing divergent control elements . Each vector contains a pair of promoterless genes {encoding beta-galactosidase (lacZ), alkaline phosphatase (phoA), and bacterial luciferase (luxAB)} arranged in an antiparallel fashion and separated by a large intervening multiple cloning site . The vectors permit direct detection of promoter activity on indicator plates after transformation . Cloned promoters are selected based on production of coloured products in the case of lacZ and phoA, and by the emission of light in the case of luxAB . These vectors have been tested using known divergent promoter elements from pBR322 and Pseudomonas phage D3.

J Biol Chem, 1990 May 25, 265(15), 8636 - 41
Involvement of denaturation-like changes in Pseudomonas exotoxin a hydrophobicity and membrane penetration determined by characterization of pH and thermal transitions . Roles of two distinct conformationally altered states; Jiang JX et al.; Previous investigators have shown that exotoxin A undergoes a conformational switch to a hydrophobic state at low pH . This change appears to play a role in exotoxin A entry into cells by facilitating its penetration of the membranes of acidic organelles . We have examined the effects of pH, temperature, and denaturants in order to define the role of conformational changes in membrane penetration by the exotoxin . We find that two distinct low pH conformations exist . An intermediate low pH state (LI) dominates at pH 3.7-5.4 and is distinguished by blue-shifted fluorescence and weak or no hydrophobicity . The second low pH state (LII) is dominant below pH 3.7 and is characterized by red shifted fluorescence and strong hydrophobicity . LI is a folded state as judged by its spectroscopic properties and the observation that it undergoes distinct and cooperative thermal and denaturant induced unfolding transitions . LII appears to be more like a denatured state, as it shows no cooperative thermal or denaturant induced transitions and has spectroscopic properties very similar to exotoxin A that has been thermally denatured at pH 7 . Exotoxin A in the LII state strongly binds detergent micelles and binds and inserts into model membranes . Therefore, denaturation-like conformational changes appear to play an important role in membrane insertion . The pH of the transition to a membrane-inserting state is influenced by the composition of the model membranes and is close to pH 5 in the presence of vesicles containing a phosphatidylglycerol/phosphatidylcholine mixture . These vesicles probably promote formation of the LII state via mass action effects . The implication of these results for membrane penetration and translocation of proteins without apparent hydrophobic regions, such as exotoxin A, is discussed.

Carbohydr Res, 1990 May 15, 199(1), 77 - 82
The structure of the acidic exopolysaccharide of Pseudomonas marginalis strains PF-05-2 and PM-LB-1; Osman SF et al.; The structure of an acidic exopolysaccharide of two strains of Pseudomonas marginalis, a bacterium which causes soft rots of various vegetables, has been determined to consist of a repeating unit of: ----4) beta-D-Manp-(1----3)alpha-D-Glcp-(1----4)alpha-L-Rhap-(1- . The glucose is pyruvated at O-4 and O-6 and the mannose is acetylated at either O-2 or O-3.

J Chromatogr, 1990 May 11, 506, 327 - 34
Peptide behaviour and analysis on a chemically stable C18-bonded vinyl alcohol copolymer column with alkaline and acidic eluents; Uchida T et al.; C18-bonded vinyl alcohol copolymer (ODP) gel showed no weight loss or decrease in column efficiency for alkyl alcohols after being immersed in aqueous solutions of pH 2 and 10 at 50 degrees C for 48 h, and only a 1% weight loss and a slight decrease in alkyl alcohol retention volumes after similar immersion at pH 13 . The chemical stability of the ODP gel was further demonstrated in analyses of acidic, neutral and basic peptides on an ODP column with eluents of pH 3-10, which showed that the peptides differ considerably in the sensitivity of their retention behaviour to eluent pH, even though hydrophobic interaction invariably appeared to be the main retention mechanism . The ODP column was therefore applied to the analysis and alignment of lysilendopeptidase (LEP) peptides derived from reduced and S-carboxymethylated carboxyl proteinase (Rcm-P-CP) of Pseudomonas sp . 101 . One acid-soluble and seven alkaline-soluble LEP peaks were found in analyses on the ODP column using acidic and alkaline eluents, respectively . The chymotryptic peptides of Rcm-P-CP were first separated on an ODS column with an acidic eluent, and the eight eluates which contained lysine residue, as determined by amino acid analysis, were then analysed on the ODP column with an alkaline eluent, resulting in a further separation of each into several peaks and thus in the recovery of fractions of pure peptides . The LEP peptide alignment was then determined by overlapping the sequences of the chymotryptic peptides with the C- and N-terminal regions of the LEP peptides.

Int J Food Microbiol, 1990 May, 10(3-4), 331 - 42
Inability of a bacteriophage pool to control beef spoilage; Greer GG et al.; The biological control of beef spoilage, with a bacteriophage (phage) pool, was evaluated under simulated retail conditions . A pool of seven phages was selected with the potential to lyse 78% of 86 Pseudomonas test strains . Subsequent host range studies with 1023 pseudomonads from three meat species (beef, pork, lamb) and five abattoirs showed that 585 (57.2%) isolates were susceptible to the phage pool . Depending on bacterial origin, bacterial sensitivity to lysis by the phage pool varied from 25 to 72% . When added to ribeye steaks, the phage pool produced a significant reduction in Pseudomonas growth but this was not sufficient to produce any significant effect upon the retail shelf life of beef . The inability of phages to control beef spoilage was not attributed to a loss of phage virulence since sufficient densities (log pfu/cm2 = 5 to 6) of virulent phage could be re-isolated from beef, 14 days after treatment . It was concluded that the efficacy of the current phage pool was limited by a narrow range of specificity.

Appl Environ Microbiol, 1990 May, 56(5), 1279 - 85
Phenol and trichloroethylene degradation by Pseudomonas cepacia G4: kinetics and interactions between substrates; Folsom BR et al.; Intact cells of Pseudomonas cepacia G4 completely degraded trichloroethylene (TCE) following growth with phenol . Degradation kinetics were determined for both phenol, used to induce requisite enzymes, and TCE, the target substrate . Apparent Ks and Vmax values for degradation of phenol by cells were 8.5 microM and 466 nmol/min per mg of protein, respectively . At phenol concentrations greater than 50 microM, phenol degradation was inhibited, yielding an apparent second-order inhibitory value, KSI, of 0.45 mM as modeled by the Haldane expression . A partition coefficient for TCE was determined to be 0.40 +/- 0.02, {TCEair}/{TCEwater}, consistent with Henry's law . To eliminate experimental problems associated with TCE volatility and partitioning, a no-headspace bottle assay was developed, allowing for direct and accurate determinations of aqueous TCE concentration . By this assay procedure, apparent Ks and Vmax values determined for TCE degradation by intact cells were 3 microM and 8 nmol/min per mg of protein, respectively . Following a transient lag period, P . cepacia G4 degraded TCE at concentrations of at least 300 microM with no apparent retardation in rate . Consistent with Ks values determined for degradation, TCE significantly inhibited phenol degradation.

J Bacteriol, 1990 May, 172(5), 2704 - 9
Molecular cloning of the protocatechuate 4,5-dioxygenase genes of Pseudomonas paucimobilis; Noda Y et al.; We determined the nucleotide sequence of a 1.9-kilobase fragment of Pseudomonas paucimobilis SYK6 chromosomal DNA that included genes encoding protocatechuate 4,5-dioxygenase, the enzyme responsible for the aromatic ring fission of protocatechuate . Two open reading frames of 417 and 906 base pairs were found that had no homology with previously reported sequences, including those encoding protocatechuate 3,4-dioxygenase . Since both open reading frames were indispensable for the enzyme activity, they should encode the subunits of protocatechuate 4,5-dioxygenase . We named these genes ligA and ligB . Protocatechuate 4,5-dioxygenase was efficiently expressed in Escherichia coli with the aid of the lac promoter, and the polypeptides of the ligA and ligB gene products were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid sequencing.

J Biochem (Tokyo), 1990 May, 107(5), 714 - 7
The methanol-oxidizing system of Methylobacterium extorquens AM1 reconstituted with purified constituents; Mukai K et al.; The electron transport system (with cytochrome aa3) coupled to the oxidation of methanol in Methylobacterium extorquens AM1 (former Pseudomonas AM1) was reconstituted with highly purified constituents of the system . A mixture of 2.7 microM methanol dehydrogenase, 3.2 microM cytochrome cH, and 71 nM cytochrome c oxidase (= cytochrome aa3) consumed oxygen at a lower rate in the presence of methanol, while its activity was enhanced 3-fold by the addition of 1.4 microM cytochrome cL (74 mol of O2 consumed/mol of heme a of cytochrome c oxidase per min) . Further addition of amicyanin to the above mixture did not affect the activity . Although ammonium ion greatly activated the activity of methanol dehydrogenase, the ion had little effect on the oxygen consumption activity of the above mixture . On the basis of the results obtained in the present study, an electron transport system is proposed for the oxidation of methanol in M . extorquens AM1.

J Immunol, 1990 May 1, 144(9), 3417 - 23
Selective elimination in vitro of alloresponsive T cells to human transplantation antigens by toxin or radionuclide conjugated anti-IL-2 receptor (Tac) monoclonal antibody; Kozak RW et al.; The human allogeneic mixed lymphocyte reaction is the in vitro correlate of graft rejection . Cytotoxic effector cells generated during an allogeneic mixed lymphocyte reaction were previously shown to express the human p55 IL-2 receptor subunit, whereas resting cells do not express this receptor peptide . In this study, we asked whether Pseudomonas exotoxin or bismuth-212 (an alpha-particle emitting radionuclide) coupled to the anti-IL-2 receptor mAb, anti-Tac, were able to selectively eliminate alloresponsive cells generated during an allogeneic mixed lymphocyte reaction . After assembly, anti-Tac immunoconjugates retained their binding integrity, specificity, and selectivity . Deletion of alloresponsive cells was shown by the removal of alloproliferating cells as assessed by quantitating cell recovery and by measurement of thymidine incorporation into newly synthesized DNA . Both toxin and radionuclide immunoconjugates eliminated established cytotoxic effector cells generated in an allogeneic mixed lymphocyte reaction, while leaving intact the PHA-inducible mitogenic response of the nonactivated cells . The addition of excess anti-Tac blocked all of the effects of these cytotoxic reagents . The therapeutic reagents in vitro were most effective when added just prior to the peak of the alloproliferative response, when receptor expression would be close to maximum . Thus, anti-Tac conjugated either with toxin or radionuclide is effective in vitro in specifically eliminating cytotoxic effector cells.

Antonie Van Leeuwenhoek, 1990 May, 57(4), 223 - 36
Numerical taxonomy of fluorescent Pseudomonas associated with tomato roots; Stenstrom IM et al.; The phenetic taxonomy of 110 fluorescent bacterial strains, isolated from the roots of tomatoes and other plants was numerically studied through 97 features including 69 assimilation tests . Thirty-two reference strains of various Pseudomonas spp . were additionally included . The strains clustered into 16 clusters at the 74% similarity level when using Jaccard similarity coefficients . Almost all field strains belonged to the P . fluorescens/P . putida-complex while none clustered with P . syringae and allied bacteria . The biovar II branch, as well as the newly described biovar VI of P . fluorescens, made up 55% and 20% respectively, of the field strains; two % were allocated to P . fluorescens biovar I and three % to biovar IV . Eleven % of the root associated strains were designated P . putida; six strains were biovar A, three strains biovar B while four strains could not be referred to any known biovar . The continuum within the P . fluorescens/P . putida-complex as well as the taxonomic status of the six biovars of P . fluorescens and the three biovars of P . putida are discussed.

Infect Immun, 1990 May, 58(5), 1415 - 20
Low pH-induced changes in Pseudomonas exotoxin and its domains: increased binding of Triton X-114; Idziorek T et al.; Pseudomonas exotoxin (PE), which is composed of three structural domains, is a 66-kilodalton protein secreted by P . aeruginosa that is cytotoxic for mammalian cells . After binding to cell surface receptors and internalization into low-pH endocytic vesicles, PE or an active fragment kills mammalian cells by translocating across an intracellular membrane to the cytoplasm and shutting down protein synthesis . To investigate possible conformational changes associated with the translocation process, full-length PE or recombinant proteins containing the PE cell recognition domain, translocation domain, enzymatic domain, or translocation plus enzymatic domains were incubated with Triton X-114 at pH values ranging from 3.0 to 7.0 . The truncated forms used were intact domains that had been expressed in Escherichia coli and subsequently purified . Previous studies (K . Sandvig and J . O . Moskaug, Biochem . J . 245:899-901, 1987) had shown that full-length PE bound more Triton X-114 at a low pH than at a physiologic pH . Therefore, we investigated whether this increased binding was due to a global change in PE or a change within a particular domain . Results showed that all the truncated toxin proteins displayed a similar pH-dependent entry into the detergent phase as native PE, with a transition point of 4.2 for PE and 4.4 to 4.5 for the truncated toxins . The isoelectric points of the recombinant proteins were measured and indicate that, at a low pH (5.0), the cell recognition domain bears a net positive charge, the translocation domain bears a net negative charge, and the enzymatic domain bears no charge . The results suggest that upon acidification in the endosome, PE becomes globally hydrophobic and is converted into a translocation-competent form.

J Hosp Infect, 1990 May, 15(4), 383 - 8
Pseudomonas paucimobilis bacteraemia associated with haemodialysis; Calubiran OV et al.; We report a case of Pseudomonas paucimobilis bacteraemia where the source was presumed to be related to haemodialysis . Previous reports have identified this organism as a pathogen in a variety of disease states including bacteraemia of unknown origin and in patients undergoing peritoneal dialysis.

Mol Pharmacol, 1990 May, 37(5), 631 - 8
Pseudomonas exotoxin A prevents beta-adrenoceptor-induced upregulation of Gi protein alpha-subunits and adenylyl cyclase desensitization in rat heart muscle cells; Reithmann C et al.; Exposure of rat heart muscle cells to noradrenaline (1 microM) for 48 hr led to a decrease in the number of beta 1-adrenoceptors of 50% and a concomitant decrease in adenylyl cyclase stimulation by isoprenaline and forskolin of about 60 and 30%, respectively . In addition, the levels of two inhibitory guanine nucleotide-binding protein (Gi protein) alpha-subunits (Gi alpha 40 and Gi alpha 41) were increased in membranes of noradrenaline-treated cells . Evidence is presented that noradrenaline induces this increase by activation of beta-adrenoceptors . First, the noradrenaline action was mimicked by the beta-adrenoceptor agonist isoprenaline . Second, beta-adrenoceptor blockade by timolol but not alpha-adrenoceptor blockade by prazosin prevented the noradrenaline-induced up-regulation of Gi alpha proteins . Furthermore, timolol but not prazosin abolished the noradrenaline-induced down-regulation of beta 1-adrenoceptors and the decreases in receptor-dependent (isoprenaline) and -independent (forskolin) adenylyl cyclase stimulation . The specific protein synthesis inhibitor Pseudomonas exotoxin A was used to study whether the noradrenaline-induced up-regulation of Gi alpha subunits depends on increased synthesis of these proteins . This toxin inhibits peptide chain elongation by ADP-ribosylating elongation factor 2 . Treatment of rat heart muscle cells with Pseudomonas exotoxin A (1 ng/ml) completely prevented the noradrenaline-induced increase in Gi alpha proteins, measured by both pertussis toxin-catalyzed ADP-ribosylation and immunoblotting with anti-Gi alpha antibodies . Most importantly, Pseudomonas exotoxin A also completely prevented the noradrenaline-induced decrease in forskolin-stimulated adenylyl cyclase activity . Furthermore, the noradrenaline-induced decrease in isoprenaline-stimulated adenylyl cyclase activity was significantly attenuated by the toxin, although the down-regulation of beta 1-adrenoceptors caused by noradrenaline treatment was not affected . The data presented suggest that prolonged activation of beta-adrenoceptors in rat heart muscle cells, in addition to causing a receptor down-regulation, induces the synthesis of Gi alpha proteins, which then apparently mediate a decreased adenylyl cyclase responsiveness . The data, additionally, suggest that the synthesis of Gi alpha proteins is under control of the activity of the adenylyl cyclase system and that altered levels of these proteins may play a major role in long term regulation of signal transduction by this enzyme.

Kansenshogaku Zasshi, 1990 May, 64(5), 604 - 11
{Studies on defence effects of recombinant human granulocyte colony-stimulating factor (G-CSF) to infections . III . Protective effect on pulmonary and systemic infections of P . aeruginosa in neutropenic mice}; Tomono K et al.; In the prior two reports, we demonstrated that G-CSF induced the polarization of neutrophils by itself, and also enhanced superoxide production from neutrophils stimulated by the chemotactic peptides . In this study, we have examined the protective effect of G-CSF in vivo on Pseudomonas pneumonia and septicemia in mice . Cyclophosphamide (CY) induced severe reduction of the number of peripheral leukocytes and weakened resistance for Psuedomonas aeruguinosa infection of mice . However, in mice receiving recombinant human G-CSF four daily subcutaneous injection, the number of leukocytes, particulary neutrophils, increased more rapidly than in controls receiving saline . Moreover G-CSF enhanced a protective effect to pulmonary and systemic pseudomonas infections . When G-CSF was administered together with antibiotics, significant synergism in the protection against pulmonary infection of Pseudomonas was observed . Carrageenan treatment decreased the protective effect of G-CSF . These results suggested that the protective effect of G-CSF against P . aeruginosa infection depends not only on PMN but also another complexed host defence mechanism.

J Biol Chem, 1990 Apr 15, 265(11), 6301 - 11
Gentisate 1,2-dioxygenase from pseudomonas . Purification, characterization, and comparison of the enzymes from Pseudomonas testosteroni and Pseudomonas acidovorans; Harpel MR et al.; The 3-hydroxybenzoate inducible gentisate 1,2-dioxygenases have been purified to homogeneity from P . acidovorans and P . testosteroni, the two divergent species of the acidovorans group of Pseudomonas . Both enzymes exhibit a 40-fold higher specific activity than previous preparations and have an (alpha Fe)4 quaternary structure (holoenzyme Mr = 164,000 and 158,000, respectively) . The enzymes have different amino terminal sequences, amino acid contents, and isoelectric points . Each enzyme contains essential active site iron that is EPR silent but binds nitric oxide quantitatively to give an EPR active complex (S = 3/2), showing that the iron is Fe2+ with coordination sites for exogenous ligands . The EPR spectra of these complexes are altered uniquely for each enzyme when gentisate is bound . This suggests that substrate binds to or near the iron and shows that the substrate-iron interactions of each enzyme are subtly different . The kinetic parameters for turnover of gentisate by the enzymes are nearly identical (kcat/Km = 4.3 x 10(6) s-1 M-1) . Both enzymes cleave a wide range of gentisate analogs substituted in the 3 or 4 ring position, although at reduced rates relative to gentisate . Of the two enzymes, P . testosteroni gentisate 1,2-dioxygenase exhibits substantially lower kcat/Km values for the turnover of these compounds . Evidence for both steric and electronic substituent effects is obtained . In accord with the results of Wheelis et al . (Wheelis, M . L., Palleroni, N . J., and Stanier, R . Y . (1967) Arch . Mikrobiol . 59, 302-314), 3-hydroxybenzoate is shown to be metabolized by P . acidovorans through the gentisate pathway, and gentisate 1,2-dioxygenase is the only ring cleavage dioxygenase induced . In contrast, 3-hydroxybenzoate is metabolized by P . testosteroni exclusively through the protocatechuate pathway utilizing protocatechuate 4,5-dioxygenase, although gentisate 1,2-dioxygenase is coinduced . Growth of P . testosteroni on 3-O-methylbenzoate or 5-O-methylsalicylate is shown to result in a approximately 10-fold increase in the amount of gentisate 1,2-dioxygenase relative to protocatechuate 4,5-dioxygenase . Together, these results suggest that induction of gentisate 1,2-dioxygenase by 3-hydroxybenzoate in P . testosteroni may be adventitious and that this enzyme may function in fundamentally different metabolic pathways in the two related Pseudomonas species.

J Biol Chem, 1990 Apr 5, 265(10), 5531 - 9
Pseudomonas cepacia 2,2-dialkylglycine decarboxylase . Sequence and expression in Escherichia coli of structural and repressor genes; Keller JW et al.; A 3969-base pair PstI-PstI fragment of Pseudomonas cepacia DNA containing the gene for the pyridoxal 5'-phosphate dependent 2,2-dialkylglycine decarboxylase (pyruvate) (EC 4.1.1.64) was cloned in Escherichia coli . The insert was sequenced by the dideoxy method using nested deletions from both ends, revealing a central 1302-base pair region that codes for the decarboxylase subunit . The recombinant enzyme was expressed in E . coli, purified to homogeneity, and sequenced at the amino terminus . Also, a cofactor-labeled active site peptide was sequenced . The carboxyl terminus of the deduced amino acid sequence is homologous with the carboxyl terminus of mammalian ornithine aminotransferase; the active site sequence is similar to the active site sequences of several other aminotransferases . No homologies with known decarboxylase sequences could be found . Expression of the decarboxylase gene is negatively controlled by a 687-nucleotide sequence upstream of and diverging from the structural gene . Expression is induced by S-isovaline, 2-methylalanine, and D-2-aminobutanoic acid, but not by glycine, D- or L-alanine, L-2-aminobutanoic acid, R-isovaline, or other alkyl amino acids.

J Gen Microbiol, 1990 Apr, 136 ( Pt 4), 607 - 13
Molecular cloning and expression of a novel catechol 2,3-dioxygenase gene from the benzoate meta-cleavage pathway in Azotobacter vinelandii; Keil H; Azotobacter vinelandii strain 206 degrades benzoate via the meta-cleavage pathway . In a genomic library derived from this organism a clone was obtained which carried and expressed the gene for the third enzyme in this pathway, catechol 2,3-dioxygenase (EC 1.13.11.2), on a 5.9 kb SalI restriction fragment . The structural gene was more precisely mapped on an internal 1.6 kb EcoRI fragment which, after insertion into expression vectors, directed the synthesis of a 33 kDa polypeptide . The gene showed very little or no homology with isofunctional genes derived from Pseudomonas . Comprehensive substrate specificity analysis showed significant differences between the specific activities obtained from the cloned gene product and extracts derived from Azotobacter itself.

Rinsho Ketsueki, 1990 Apr, 31(4), 474 - 8
{Phenytoin induced agranulocytosis: direct and T cell-mediated mechanisms}; Matsuzaki M et al.; A 57-year-old male was admitted to our hospital because of high fever and dyspnea . He had convulsive attacks after the operation for subarachnoid hemorrhage . He received an anticonvulsant, Hydantol F (Phenytoin + Phenobarbital), for 20 days before hospitalization . The white cell count on admission was 900/microliters without any neutrophils . Pseudomonas was demonstrated from blood cultures . After the anticonvulsant was discontinued and potent antibiotics were administered, his symptoms improved . Neutrophils appeared in the peripheral blood . We investigated the effect of phenytoin on the granuloid precursors using in vitro colony assay (CFU-C) after recovery from agranulocytosis . There was substantial decrease in CFU-C colony numbers when phenytoin or peripheral T-lymphocytes incubated with phenytoin was added in the culture . The addition of sera from acute phase did not affect the colony formation . These results suggested that direct toxic effect of the drug and cellular immunity played an important role in phenytoin induced agranulocytosis.

Ann Rheum Dis, 1990 Apr, 49(4), 258 - 9
Septic arthritis caused by treatment resistant Pseudomonas cepacia; Matteson EL et al.; A case of septic arthritis of the knee caused by Pseudomonas cepacia following an intraarticular corticosteroid injection in a patient with a history of osteoarthritis is presented . This is the second report of this agent causing infection in a diarthrodial joint, which proved difficult to eradicate despite in vitro antibiotic sensitivity.

Jpn J Cancer Res, 1990 Apr, 81(4), 396 - 402
Tumor necrosis factor changes sensitivity of differentiation of mouse leukemia M1 cells by lipopolysaccharide; Nakaya K et al.; A clone of mouse leukemia M1 cells was induced to differentiate by lipopolysaccharide (LPS) (LPS-sensitive clone) while another clone of the same cells was resistant (LPS-resistant clone) . LPS and lipid A preparations from Pseudomonas diminuta and Pseudomonas vesicularis were as active as Escherichia coli LPS in the induction of differentiation of the LPS-sensitive clone . Synthetic lipid A precursor Ia (compound 406), which has no interleukin 1 (IL-1)-inducing activity toward monocytes, had strong differentiation-inducing activity toward the LPS-sensitive clone . The combined treatment of the LPS-sensitive clone with LPS and recombinant tumor necrosis factor (rTNF) did not further increase the degree of differentiation induced by LPS alone . By contrast, the LPS-resistant clone was markedly induced to differentiate by LPS in the presence of rTNF . Combined treatment of the LPS-resistant clone with LPS and other cytokines such as recombinant IL-1 alpha, recombinant granulocyte colony-stimulating factor, and interferon-gamma was not effective in inducing marked synergistic differentiation . These results raise the possibility that rTNF changes the sensitivity of M1 cells to induction of differentiation by LPS.

Biochimie, 1990 Apr, 72(4), 285 - 9
Beta-hydroxysteroid dehydrogenase: activity in microemulsion and extraction from Pseudomonas testosteroni cells with microemulsion; Lee KM et al.; The stability of purified beta-hydroxysteroid dehydrogenase activity measured as a function of time was good in buffered cationic and non-ionic microemulsions . The use of 1-pentanol and 1-hexanol in place of 1-butanol as cosurfactant gave increased activity and stability . The NAD+ Michaelis constant was 0.22 mM in buffer and 3.5 mM in waterpool concentration in microemulsion . Proteins, among them beta-hydroxysteroid dehydrogenase, were extracted from Pseudomonas testosteroni with cationic microemulsion, thus indicating that microemulsions may be utilized in protein release from cells.

Arch Ophthalmol, 1990 Apr, 108(4), 504 - 8
Microsporidial keratoconjunctivitis in acquired immunodeficiency syndrome; Friedberg DN et al.; We describe three patients with acquired immunodeficiency syndrome who presented with a bilateral coarse superficial epithelial keratitis due to infection with the protozoal parasite Microspora, Encephalitozoon cuniculi . Despite the extent of the corneal surface disease, conjunctival inflammation was minimal . Visual acuity ranged from 20/20 to 20/200 . In one patient, the keratitis was complicated by the development of a surface defect with secondary Pseudomonas species infection . All patients had a history of exposure to household pets . Standard cultures were negative . Diagnosis was established in two of the three cases based on characteristic appearance of the protozoan in conjunctival scrapings . Electron microscopy of a conjunctival biopsy specimen in one patient confirmed the species . No recognized effective treatment is available for this infection.

Ann Surg, 1990 Apr, 211(4), 399 - 405
Bacterial translocation and intestinal atrophy after thermal injury and burn wound sepsis; Jones WG 2nd et al.; Bacterial translocation (BT) occurs after thermal injury in rodents in association with intestinal barrier loss . Infection complicating thermal injury may also affect the intestine producing bowel atrophy . To study these relationships, Wistar rats received either 30% scald followed by wound inoculation with Pseudomonas; 30% scald with pair feeding to infected animals; or sham injury as controls . On days 1, 4, and 7 after injury animals were killed with examination of the bowel and culture of the mesenteric lymph nodes (MLN), livers, spleens, and blood . All burned animals demonstrated BT to the MLN on day 1 after injury, but only burn-infected animals had continued BT on days 4 and 7, with progression of BT to the abdominal organs and blood . Burn injury and infection also resulted in significant atrophy of small bowel mucosa temporally associated with continued BT . Thus injury complicated by infection results in prolonged and enhanced bacterial translocation, perhaps due to failure to maintain the mucosal barrier.

Int J Biol Macromol, 1990 Apr, 12(2), 92 - 101
Bacterial polyesters containing branched poly(beta-hydroxyalkanoate) units; Fritzsche K et al.; Pseudomonas oleovorans was grown on mixtures of methyloctanoates with n-octanoate . Polymers were also obtained from organisms grown on pure 7-methyloctanoate, but not from pure 5- or 6-methyloctanoate . The polyesters obtained from 7-methyloctanoate and from its mixtures with n-octanoate contained units with the methyl branches in the pendant group, as did the copolymers from the mixtures of 5- and 6-methyloctanoate with n-octanoate . The methyl branched repeating units contained two diastereomers, and the 13C-n.m.r . spectra of these polymers indicated that the 5-methyloctanoate units had a higher content of one of the two isomers, but not in the 6-methyloctanoate units . The weight average molecular weights of the copolyesters produced were in the range of 220,000 to 410,000, with Mw/Mn ratios of 1.7 to 1.9.

Int J Biol Macromol, 1990 Apr, 12(2), 85 - 91
Production of unsaturated polyesters by Pseudomonas oleovorans; Fritzsche K et al.; Pseudomonas oleovorans was grown separately on 3-hydroxy-6-octenoic acid and 3-hydroxy-7-octenoic acid as the only carbon source and under ammonium nutrient-limiting conditions to produce storage polyesters . The polyesters produced contained mainly unsaturated C8 units . Small amounts of both the saturated and the unsaturated C6 units were also present, but only about 1% of the saturated 3-hydroxyoctanoate units was detected . The polyester obtained from 3-hydroxy-6-octenoic acid, which was a mixture of the cis and trans isomers, also contained units with cis and trans double bonds . The weight average molecular weights of the polymers produced were in the range of 339,000-383,000 as determined by g.p.c . relative to polystyrene, with Mw/Mn ratios of 1.8-2.1 . The mechanism of PHA formation from n-octene previously reported is discussed in relation to the present results, and the two were found to be in good agreement.

Agric Biol Chem, 1990 Apr, 54(4), 921 - 6
Purification and some properties of endo-1,3-beta-D-xylanase from Pseudomonas sp . PT-5; Yamaura I et al.; An endo-1,3-beta-D-xylanase (1,3-beta-D-xylan xylanohydrolase, EC 3.2.1.32) was purified from the culture fluid of Pseudomonas sp . PT-5 by ammonium sulfate fractionation, DEAE-Sepharose CL-6B, Toyopearl HW-50S, and Butyl-Toyopearl 650 M column chromatography . The purified enzyme gave a single band on polyacrylamide gel disc electrophoresis and its molecular weight was 35,000 by SDS-polyacrylamide gel electrophoresis . The enzyme was stable from pH 5.5 to 8.0 and had its maximum activity at pH 7.5 . The enzyme rapidly reduced the viscosity of glycol beta-1,3-xylan solutions and produced xylose and xylooligosaccharides from seaweed beta-1,3-xylan . The enzyme activity was greatly inhibited by Hg2+, SDS, ethylenediamine tetraacetic acid (EDTA), and N-bromosuccinimide (NBS).

J Ind Microbiol, 1990 Apr-May, 5(2-3), 79 - 84
Bacterial conjugation between Escherichia coli and Pseudomonas spp . donor and recipient cells in soil; Berg G et al.; Experiments conducted in microcosms containing loam soil samples inoculated with either E . coli or Pseudomonas spp . donor and recipient cells showed that bacterial cells survived and conjugated over a 24-h incubation period . E . coli transconjugants were detected 6 h after donor and recipient strains were introduced into sterile soil samples . In non-sterile soil samples, transconjugants were detected between 8 and 24 h incubation . Pseudomonas transconjugants were recovered from sterile soil samples between 6 and 12 h after their introduction and as early as 2 h in non-sterile soil . The results show that genetic interactions occur in non-sterile soil in relatively short periods of time at relatively high transfer frequencies (10(-3) to 10(-4} . Studies on genetic interactions in soil are becoming necessary in risk assessment/environmental impact studies prior to the release of genetically engineered or modified organisms into uncontained environments.

J Mol Biol, 1990 Mar 5, 212(1), 135 - 42
Rubredoxin reductase of Pseudomonas oleovorans . Structural relationship to other flavoprotein oxidoreductases based on one NAD and two FAD fingerprints; Eggink G et al.; The oxidation of alkanes to alkanols by Pseudomonas oleovorans involves a three-component enzyme system: alkane hydroxylase, rubredoxin and rubredoxin reductase . Alkane hydroxylase and rubredoxin are encoded by the alkBFGHJKL operon, while previous studies indicated that rubredoxin reductase is most likely encoded on the second alk cluster: the alkST operon . In this study we show that alkT encodes the 41 x 10(3) Mr rubredoxin reductase, on the basis of a comparison of the expected amino acid composition of AlkT and the previously established amino acid composition of the purified rubredoxin reductase . The alkT sequence revealed significant similarities between AlkT and several NAD(P)H and FAD-containing reductases and dehydrogenases . All of these enzymes contain two ADP binding sites, which can be recognized by a common beta alpha beta-fold or fingerprint, derived from known structures of cofactor binding enzymes . By means of this amino acid fingerprint we were able to determine that one ADP binding site in rubredoxin reductase (AlkT) is located at the N terminus and is involved in FAD binding, while the second site is located in the middle of the sequence and is involved in the binding of NAD or NADP . In addition, we derived from the sequences of FAD binding reductases a second amino acid fingerprint for FAD binding, and we used this fingerprint to identify a third amino acid sequence in AlkT near the carboxy terminus for binding of the flavin moiety of FAD . On the basis of the known architecture and relative spatial orientations of the NAD and FAD binding sites in related dehydrogenases, a model for part of the tertiary structure of AlkT was developed.

Farmakol Toksikol, 1990 Mar-Apr, 53(2), 55 - 7
{The effect of stimulants of immunity on anti-infective resistance and on the activity of the liver monooxygenase system}; Sibiriak SV et al.; The effect of some immunostimulants (bacterial lipopolysaccharide prodigiosan, active thymic peptide T-activin, synthetic compound levamisole) on the anti-infection resistance and metabolic function of the liver (hexobarbital sleeping-time) was studied on noninbred male mice . It was found that when administered in doses and under schedules that protected mice against lethal infection (Pseudomonas pyocyanea, i.p.) prodigiosan and levamisole inhibited metabolism of hexobarbital . T-activin was inactive in both tests . The possible mechanism of correlation is discussed.

Ukr Biokhim Zh, 1990 Mar-Apr, 62(2), 40 - 7
{Isolation and partial characteristics of biopolymers in the outer membrane of Pseudomonas syringae}; Mykhal's'kyi LO et al.; Conditions are developed for obtaining extracellular biopolymers (EBP), water (W), salt-EDTA (SE) and salt-detergent (SD) extracts of Pseudomonas syringae pv . atrofaciens IMV K-1025 . The fraction of unsoluble LPS is effectively precipitated from samples by ultracentrifugation (UC) . The fractions enriched by proteins and soluble LPS are salted-out from the UC supernatants by ammonium sulphate, that is confirmed by the chemical analysis, electrophoresis in the PAAG-NDS and in ELISA with the monoclonal antibodies against the homologous LPS, a comparative analysis of electrophoretic spectra of UC supernatants of EBP, W and SE extracts has shown their great similarity in the composition rather than in the specific content of basic and minor polypeptides . Nine polypeptides of the major membrane of Pseudomonas syringae pv . atrofaciens IMV K-1025 having the molecular weights of 15.5, 30.0, 34.0, 36.0, 38.0, 41.0, 42.0, 43.0 and 65.0 kDa are identified as major ones.

Bioorg Khim, 1990 Mar, 16(3), 345 - 57
{NAD-dependent formate dehydrogenase of methylotrophic bacteria Pseudomonas sp . 101 . III . Comparative analysis}; Lamzin VS et al.; The comparative analysis of the primary and tertiary structures of NAD-dependent bacterial formate dehydrogenase (FDH) from methylotrophic bacterium Pseudomonas sp . 101 and a number of structurally characterized NAD-dependent dehydrogenases were performed . FDH has a highly conservative fold of the coenzyme binding domain . Position of the symmetry axis in the FDH molecule relative to the beta-sheets of its coenzyme binding domain with the respective sequences of the other NAD-dependent enzymes was performed on the basis of the spatial homology between these structures . Only one of the three amino acid residues previously thought to be conserved in the coenzyme binding domains of NAD-dependent dehydrogenases is preserved in the FDH molecule (Asp-221) . Two glycine residues found in all previously studied dehydrogenases are substituted in FDH by Ala-198 and Pro-256, respectively . Position of the essential thiol of FDH (Cys-255) in the protein structure was established . It is suggested that Cys-255 is situated on or near polypeptide locus taking part in the conformational changes of the protein in the course of the catalysis.

Bioorg Khim, 1990 Mar, 16(3), 336 - 44
{NAD-dependent formate dehydrogenase of methylotrophic bacteria Pseudomonas sp . 101 . II . Enzyme conformation at 3.0 A resolution}; Lamzin VS et al.; Three heavy atom isomorphous derivatives were used for the X-ray analysis of the holo form of NAD-dependent bacterial formate dehydrogenase (ternary complex enzyme-NAD-azide) at 3.0 A resolution . The enzyme subunit contains a catalytic and a coenzyme binding domain, with the active centre and the coenzyme binding site in the cleft between the domains . The polypeptide chain's fold and the position of 393 C alpha-atoms were determined . The secondary structure of the formate dehydrogenase was resolved . The structure of the NAD-binding domain is shown to be similar to that of other NAD-dependent enzymes.

Bioorg Khim, 1990 Mar, 16(3), 324 - 35
{NAD-dependent formate dehydrogenase from methylotrophic bacteria Pseudomonas sp . 101 . I . Amino acid sequence}; Popov VO et al.; The primary structure of NAD-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp . 101 is determined . The enzyme is composed of two identical subunits, each comprising 393 amino acid residues, and has a molecular weight of 43.1 kD . To elucidate the protein's amino acid sequence, four types of digestion were used: cyanogen bromide cleavage at methionine residues, endoproteinase Lys-C digestion at lysine residues, endoproteinase Glu-C cleavage at glutamic acid residues, and tryptic digestion . The peptides obtained were purified to homogeneity and characterized.

Ann Acad Med Singapore, 1990 Mar, 19(2), 290 - 4
Necrotising fasciitis in leukaemic children; Lou J et al.; This is a report of necrotising subcutaneous infection in the perineum of three young girls with acute lymphoblastic leukaemia (ALL) . This occurred during induction therapy when they were granulocytopenic . Pseudomonas aeroginosa was isolated from both blood and affected sites . With aggressive chemotherapy and extensive surgical debridement, two patients were salvaged but one perished from the septicaemia . Necrotizing Fasciitis is an uncommon progressive infection of the subcutaneous tissue . It occurs more frequently in adults following surgery or trauma and rarely arises spontaneously . In the paediatric literature, reports are few and mainly in healthy children occurring post-operatively . There was only one case of this occurring in a boy with Aplastic anaemia . Early recognition and aggressive therapy helps to improve the morbidity and mortality of this devastating condition.

Mol Immunol, 1990 Mar, 27(3), 273 - 82
Immunotoxins of Pseudomonas exotoxin A (PE): effect of linkage on conjugate yield, potency, selectivity and toxicity; Morgan AC Jr et al.; Conjugates of monoclonal antibodies and Pseudomonas exotoxin A (PE) were formed with disulfide or thioether bonds . Thioether conjugates which formed with succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) modified PE and reduced antibody formed with an 80% yield of equimolar conjugate within 30 min with an offering of one to one (toxin:antibody) . The efficiency and kinetics of thioether formation were much higher with SMCC than with other maleimide reagents as well as more efficient than disulfide linkers . Thioether linkage resulted in immunotoxin consistently more potent and more selective in vitro than disulfide bonded conjugate . Thioether bonded conjugates also proved to have other favorable in vivo properties compared to disulfide conjugates: (1) a longer half-life in serum; (2) increased tumor localization; and (3) reduced toxicity . Toxicity of thioether linked holotoxin conjugates was directed at the liver hepatocyte but was easily monitored by serum liver enzymes . The conjugates are currently undergoing clinical evaluation for treatment of ovarian cancer with intraperitoneal administration . Research is ongoing to further decrease residual toxicity without reducing the potency of the conjugate.

Vet Microbiol, 1990 Mar, 22(1), 69 - 78
Selective extraction of outer-membrane proteins from membrane complexes of Pseudomonas maltophila by chloroform-methanol; Chin J et al.; An organic phase partitioning method is described for the selective purification of outer-membrane proteins (OMPs) from the total membrane complex of the opportunistic human and sheep pathogen, Pseudomonas maltophila . SDS-PAGE analysis confirmed that OMPs purified by chloroform-methanol treatment of the total membrane complex were not only identical to OMPs extracted from outer-membrane vesicles separated by sucrose gradient density centrifugation, but also possessed little or non-detectable levels of inner-membrane contaminants . Further analysis by enzyme linked immunosorbent assay (ELISA) and immunoblotting established that OMPs extracted by organic phase partitioning with chloroform-methanol retained antigenicity and serological activity indistinguishable from OMPs that were present in outer-membrane vesicles resolved by isopycnic sucrose density centrifugation of sarkosyl-treated membrane complexes.

Ann Plast Surg, 1990 Mar, 24(3), 279 - 82
Acute Pseudomonas chondritis as a sequel to ear piercing; Turkeltaub SH et al.; A patient is reported who had Pseudomonas chondritis secondary to ear piercing . The cause, pathogenesis, symptoms, and diagnosis of acute chondritis are discussed . Principles of treatment are enumerated.

Rev Infect Dis, 1990 Mar-Apr, 12(2), 173 - 80
Malignant external otitis: report on therapy with ceftazidime and review of therapy and prognosis; Johnson MP et al.; We report the treatment of 20 patients with malignant external otitis (MEO) since 1980 . Ceftazidime was used in 15 patients, with cure achieved in 11 of 12 evaluatable patients . An aminoglycoside and an antipseudomonal penicillin were used in five patients, four of whom were cured . The presentation, radiographic studies, therapy, outcome, and period of follow-up in the 20 patients are reported . The previously reported cases of MEO are also reviewed, with a focus on the changing therapy and prognosis . The frequencies of diabetes mellitus, cranial nerve deficits, and treatment failures in MEO have all declined significantly since 1985 from frequencies in earlier years . We conclude that there has been an overall improvement in the diagnosis and treatment of MEO and that monotherapy with ceftazidime shows promise against this potentially fatal pseudomonal infection.

Mol Plant Microbe Interact, 1990 Mar-Apr, 3(2), 94 - 102
Molecular characterization of avirulence gene D from Pseudomonas syringae pv . tomato; Kobayashi DY et al.; Avirulence gene D, cloned from Pseudomonas syringae pv . tomato, caused P . s . pv . glycinea to elicit a hypersensitive defense response on certain cultivars of soybean . Nucleotide sequence data for a 5.6-kb HindIII fragment containing avrD disclosed five long open-reading frames (ORFs) occurring in tandem . The phenotype conferred by avrD was expressed in P . s . pv . glycinea solely by the first of these ORFs (933 bases) that encoded a protein of 34,115 daltons . Neither a signal peptide sequence nor significant regions of hydrophobicity were present that would indicate secretion of the protein or its membrane association . Hybridization analyses revealed that some but not all P . syringae pathovars contained DNA homologous to avrD . This included weak hybridization to all tested races of P . s . pv . glycinea, although none of them express the phenotype conferred by avrD . The avrD gene occurred on an indigenous 75-kb plasmid in several P . s . pv . tomato isolates.

Mol Plant Microbe Interact, 1990 Mar-Apr, 3(2), 103 - 11
A gene from Pseudomonas syringae pv . glycinea with homology to avirulence gene D from P . s . pv . tomato but devoid of the avirulence phenotype; Kobayashi DY et al.; A gene was cloned from Pseudomonas syringae pv . glycinea that hybridized to avirulence gene D (avrD), previously cloned from P . s . pv . tomato . Unlike avrD, the hypersensitive response (HR) was not elicited when the P . s . pv . glycinea gene was reintroduced into P . s . pv . glycinea race 4 on a broad host range plasmid and the bacteria were inoculated into soybean leaves . DNA sequence data disclosed that the P . s . pv . glycinea homologue of avrD encoded a protein containing 86% identical amino acids to avrD, with substitutions distributed throughout the protein . Two ORFs immediately downstream from the avrD homologue were more similar in P . s . pv . tomato and P . s . pv . glycinea, with 98 and 99% identical amino acids . Expression of the wildtype P . s . pv . glycinea gene and recombinant genes constructed between the P . s . pv . tomato avrD gene and its P . s . pv . glycinea homologue in both Escherichia coli and P . s . pv . glycinea indicated that the P . s . pv . glycinea gene product was formed less efficiently or was less stable than was the P . s . pv . tomato protein encoded by avrD . The data indicated that the P . s . pv . glycinea homologue represents a recessive allele of the P . s . pv . tomato avrD gene which has been modified by mutation such that it does not lead to an avirulence phenotype on the normal host plant, soybean.

Gene, 1990 Mar 1, 87(1), 145 - 9
Cloning vectors, derived from a naturally occurring plasmid of Pseudomonas savastanoi, specifically tailored for genetic manipulations in Pseudomonas; Nieto C et al.; A minimal replicon of 1.8 kb isolated from a 10-kb plasmid of Pseudomonas savastanoi, pPS10, has been used to obtain a collection of small vectors specific for Pseudomonas (P . savastanoi, P . aeruginosa and P.putida) . In addition, shuttle vectors that can be established both in Pseudomonas and Escherichia coli have been constructed by adding a pMB9 replicon . The vectors permit cloning of DNA fragments generated by a variety of restriction enzymes using different antibiotic resistance markers for selection and offer the possibility to screen recombinants by insertional inactivation . This cloning system can be used to establish recombinant plasmids in Pseudomonas either at low or high copy number . pPS10 derivatives are compatible with other Pseudomonas vectors derived from broad-host-range replicons of the incompatibility groups P1, P4/Q and W . Introduction and expression of the iaaMH operon in a P . savastanoi mutant deficient in the production of indoleacetic acid has been achieved using one of these vectors.

Genetika, 1990 Mar, 26(3), 418 - 23
{Cloning of genes for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase from methylotrophic Pseudomonas in Escherichia coli}; Olekhnovich IN et al.; The genes of facultative methylotrophic bacteria Pseudomonas sp . M encoding tyrosine-sensitive and tryptophan-sensitive isoenzymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase were cloned separately on the pBR322 plasmid by complementation in Escherichia coli . Transcription of both genes in the new host was performed via the control of genes' own promoters . Cloned genes which are repressed by tyrosine and phenylalanine in their native host were expressed constitutively in E . coli.

J Bacteriol, 1990 Mar, 172(3), 1595 - 9
In vivo expression of the Pseudomonas stutzeri maltotetraose-forming amylase gene (amyP); Fujita M et al.; Northern hybridization and S1 nuclease mapping revealed that the amyP gene coding for maltotetraose-forming amylase of Pseudomonas stutzeri MO-19 is transcribed as a monocistronic mRNA of 2.0 kilobases and that the transcription start site is located 81 base pairs upstream from the first nucleotide of the initiation codon . The amyP gene was expressed weakly in Escherichia coli, and transcription started 49 base pairs downstream of the P . stutzeri MO-19 transcription start site . Synthesis of the amylase in P . stutzeri MO-19 was induced by the addition of maltose to the culture medium and was repressed by the addition of glucose . The induction by maltose was shown to be result of transcription induction of the amyP gene . In contrast, glucose did not repress transcription initiation of amyP, indicating that it controls synthesis of the enzyme, probably at the posttranscriptional level.

Agric Biol Chem, 1990 Mar, 54(3), 737 - 43
Purification and characterization of two forms of maltotetraose-forming amylase from Pseudomonas stutzeri; Nakada T et al.; Pseudomonas stutzeri MO-19 produced two active forms of extracellular maltotetraose-forming amylase . Both forms, G4-1 and G4-2, were purified to electrophoretic homogeneity . The molecular masses of G4(-1) and G4(-2) were 57 kd and 46 kd by SDS-polyacrylamide gel electrophoresis, respectively . An identical N-terminal sequence up to 20 amino acid residues and similar amino acid compositions were obtained from both forms, but different C-terminal amino acids, leucine from G4(-1) and alanine from G4(-2), were released by carboxypeptidase Y . By in vitro incubation with a culture supernatant containing protease activity, G4(-1) was converted into G4(-2) without any loss of the amylase activity . It was concluded that G4(-2) was a product derived by the limited proteolysis of G4(-1), and that the proteolysis occurred in the C-terminal region of G4-1 . G4-2 was more thermostable than G4(-1), and had a 20-fold higher Michaelis constant value for glycogen, which was 50 mg/ml against 2.3 mg/ml of G4(-1) . G4(-1) adsorbed onto raw starch granules while G4(-2) did not.

Am J Kidney Dis, 1990 Feb, 15(2), 155 - 9
A randomized prospective trial of three different regimens of treatment of peritonitis in patients on continuous ambulatory peritoneal dialysis; Chan MK et al.; A randomized prospective study was undertaken in patients on continuous ambulatory peritoneal dialysis (CAPD) to evaluate the efficacy of three different antibiotic regimens for the treatment of peritonitis . There were 39 episodes in each treatment group . Patients were treated with intraperitoneal (IP) cephalothin (250 mg/L) and tobramycin (8 mg/L) in group 1, oral ofloxacin (400 mg loading followed by 300 mg daily) in group 2, and a combination of ofloxacin (400 mg followed by 300 mg daily) and rifampicin (300 mg daily) . Treatment duration was 10 days . The average culture-positive rate was 75% . The overall cure rate was 80.6% with IP antibiotics, 78.4% with oral ofloxacin, and 81.1% with ofloxacin and rifampicin . After the exclusion of tunnel infections and episodes of peritonitis due to Pseudomonas and resistant organisms, the corresponding figures were 100%, 90.6%, and 93.7%, respectively . Side effects were minimal with IP treatment and with oral ofloxacin, but severe nausea and vomiting occurred in some cases with the combination of ofloxacin and rifampicin . It was concluded that oral ofloxacin is an acceptable first-line therapy for peritonitis in CAPD patients.

Pneumologie, 1990 Feb, 44 Suppl 1, 661 - 2
{Unilateral lung transplantation--an initial report of experiences}; Toomes H et al.; Lung transplants have been attempted since 1963 but with little success . After fundamental work by the Toronto group with improved surgical techniques and after the introduction of cyclosporin that group was able to present convincing results . Of 16 unilaterally transplanted patients with pulmonary fibrosis in the final stage, 10 are still living, one of them for now 5 years . With bilateral lung transplantation the indication was extended to cover further pulmonary diseases in the final stage, such as emphysema, bronchiectases, eosinophilic granuloma, primary pulmonary hypertension and bronchiolitis obliterans . The unilateral lung transplantation performed by us failed after initially excellent functioning, on the 9th postoperative day because of a Pseudomonas infection that had been transferred with the donor organ.

J Biochem (Tokyo), 1990 Feb, 107(2), 184 - 9
Purification of the multienzyme complex for fatty acid oxidation from Pseudomonas fragi and reconstitution of the fatty acid oxidation system; Imamura S et al.; The multienzyme complex for fatty acid oxidation was purified from Pseudomonas fragi, which was grown on oleic acid as the sole carbon source . This complex exhibited enoyl-CoA hydratase {EC 4.2.1.17}, 3-hydroxyacyl-CoA dehydrogenase {EC 1.1.1.35}, 3-oxoacyl-CoA thiolase {EC 2.3.1.16}, cis-3,trans-2-enoyl-CoA isomerase {EC 5.3.3.3}, and 3-hydroxyacyl-CoA epimerase {EC 5.1.2.3} activities . The molecular weight of the native complex was estimated to be 240,000 . Two types of subunits, with molecular weights of 73,000 and 42,000, were identified . The complex was composed of two copies each of the 73,000- and 42,000-Da subunits . The beta-oxidation system was reconstituted in vitro using the multienzyme complex, acyl-CoA synthetase and acyl-CoA oxidase . This reconstituted system completely oxidized saturated fatty acids with acyl chains of from 4 to 18 carbon atoms as well as unsaturated fatty acids having cis double bonds extending from odd-numbered carbon atoms . However, unsaturated fatty acids having cis double bonds extending from even-numbered carbon atoms were not completely oxidized to acetyl-CoA: about 5 mol of acetyl-CoA was produced from 1 mol of linoleic or alpha-linolenic acid, and about 2 mol of acetyl-CoA from 1 mol of gamma-linolenic acid . These results suggested that the 3-hydroxyacyl-CoA epimerase in the complex was not operative . When the epimerase was by-passed by the addition of 2,4-dienoyl-CoA reductase to the reconstituted system, unsaturated fatty acids with cis double bonds extending from even-numbered carbon atoms were also completely degraded to acetyl-CoA.

AIDS Res Hum Retroviruses, 1990 Feb, 6(2), 193 - 203
Selective killing of HIV-infected cells by anti-gp120 immunotoxins; Matsushita S et al.; Either ricin A chain (RAC) or Pseudomonas exotoxin (PE) was conjugated with a murine monoclonal antibody (0.5 beta) directed against an external envelope glycoprotein (gp120) of human immunodeficiency virus (HIV) . Effects of the immunotoxins produced against infected cells were evaluated . Selective inhibition of the proliferation and killing of chronically HIV infected cells were observed in the presence of the immunotoxins . To determine the feasibility of the immunotoxins against the infected cells in seropositive subjects, we attempted to detect gp120-bearing cells in peripheral blood mononuclear cells (PBM) by cytofluorography . Cells in the monocyte/macrophage region of 2 of 10 PBM samples from HIV-infected individuals were found to react with 0.5 beta (18.1% and 12.8%) . Furthermore, the cell population which was reactive with 0.5 beta was also susceptible to RAC conjugated with 0.5 beta . These results suggest that the strategy of using anti-gp120 immunotoxin to eliminate HIV-infected cells may be feasible in infected individuals.

Anal Biochem, 1990 Feb 1, 184(2), 311 - 6
A simple enzymatic method for the preparation of radiolabeled erucoyl-CoA and other long-chain fatty acyl-CoAs and their characterization by mass spectrometry; Taylor DC et al.; A simple two-step method for the biosynthesis of radiolabeled erucoyl-coenzyme A of high specific activity and other long-chain fatty acyl-coenzyme A (acyl-CoA) thioesters is reported . 1-14C-labeled erucic and oleic acids, as well as unlabeled ricinoleic and nervonic acids, were incubated at 35 degrees C with coenzyme A in the presence of ATP, MgCl2, and acyl-CoA synthetase (EC 6.2.1.3) from Pseudomonas spp . to yield the corresponding CoA thioesters . Following incubation, each thioester was purified by rapid passage through a disposable reverse-phase C18 extraction column . The overall yields were greater than 90% and the purities greater than 95%, based on the distribution of radioactivity, and chromatographic and spectral properties . Fast ion bombardment-mass spectrometry was employed to confirm the structures of the various acyl-CoAs.

Clin Otolaryngol, 1990 Feb, 15(1), 7 - 10
A double-blind, randomized, prospective trial of a topical antiseptic versus a topical antibiotic in the treatment of otorrhoea; Clayton MI et al.; The clinical efficacy was assessed of a topical antiseptic (aluminium acetate) and a topical antibiotic (gentamicin sulphate) for the initial treatment of otorrhoea . Evidence of resistant organisms developing to either treatments after 9 and 21 days was also examined . 139 affected ears were entered into the trial and of these, 102 (74%) completed the study . Improvement in the otorrhoea occurred in 68% of ears treated with gentamicin and 67% of ears treated with aluminium acetate, with no significant difference between the two treatments . No resistant organisms to aluminium acetate were encountered . Twelve gentamicin-treated ears had gentamicin-resistant organisms at presentation and one patient developed a gentamicin-resistant Pseudomonas during treatment . We therefore recommend a topical antiseptic such as aluminium acetate rather than a topical antibiotic in the initial treatment of otorrhoea on the grounds of cost, avoidance of resistance and toxicity.

Ear Nose Throat J, 1990 Feb, 69(2), 119 - 20, 122-3
Noma and noma neonatorum; Eisele DW et al.; Noma and noma neonatorum are rare gangrenous diseases that result in mutilating loss of tissue in the oronasal region . Noma usually occurs in patients between the ages of 2 and 5 years who are malnourished, have suffered a precedent illness, or are in some way immunodeficient, or all of the above . The gangrenous slough is thought to be caused by a mixed infection of oral bacterial pathogens . The disease may be fatal when it occurs in a severely debilitated patient . Noma neonatorum produces somewhat similar appearing lesions in the neonate . The infectious organism is usually Pseudomonas and the disease is generally accompanied by a life-threatening pseudomonal sepsis . Both diseases are rare in North America . Patients with noma and noma neonatorum were treated at the Children's Hospital and Medical Center, Seattle, WA . We present these cases and a literature review.

Proc Natl Acad Sci U S A, 1990 Feb, 87(3), 1066 - 70
A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins; Chaudhary VK et al.; We have devised a strategy based on polymerase chain reaction (PCR) for the rapid cloning of functional antibody genes as single-chain immunotoxins . RNA from a hybridoma producing an antibody (OVB3) that reacts with ovarian cancer cells was used as a template to make the first strand of a cDNA . Then a second strand was synthesized and amplified by using two sets of DNA primers that (i) hybridized to the ends of the light- and heavy-chain variable regions, (ii) encoded a linker peptide, and (iii) contained appropriate restriction enzyme sites for cloning . After 30 cycles of PCR, the DNA fragments containing sequences encoding the light- and heavy-chain variable regions were cloned into an Escherichia coli expression vector containing a portion of the Pseudomonas exotoxin gene . Clones encoding recombinant single-chain immunotoxins were expressed in E . coli and the protein product was assessed for its ability to bind to or kill cells bearing the OVB3 antigen . By using this approach it should be possible to rapidly clone the functional variable region sequences of many different antibodies from hybridoma RNA.

FEBS Lett, 1990 Jan 29, 260(2), 297 - 300
Mapping of the immunodominant regions of the NAD-dependent formate dehydrogenase; Bogdanova AV et al.; A panel of 4 monoclonal antibodies and 7 polyclonal antisera against NAD-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp . 101 has been obtained . The reactivity of the 37 overlapping proteolytic peptides with the monoclonal antibodies and polyclonal antisera has been studied with ELISA test . The data obtained were interpreted residing on the structural model of the formate dehydrogenase at 3 A resolution . The immunodominant regions in the formate dehydrogenase molecule and the epitopes for the monoclonal antibodies were elucidated.

J Mol Biol, 1990 Jan 20, 211(2), 373 - 82
Signal-regulator interactions . Genetic analysis of the effector binding site of xylS, the benzoate-activated positive regulator of Pseudomonas TOL plasmid meta-cleavage pathway operon; Ramos JL et al.; This study reports a genetic analysis of the interactions between a positive regulator of gene expression and its effector molecules . Transcription of the TOL plasmid meta-cleavage pathway operon is specifically stimulated by the XylS protein positive regulator either through activation of this regulator by benzoate effectors or through its hyperproduction . One xylS mutant that exhibits constitutive expression of the operon promoter has been characterized, together with six mutants encoding altered XylS proteins that recognize as effectors benzoate analogues that are non-effectors for the XylS wild-type protein . The changes in two mutant regulators are located at the N-terminal end of the protein, within a putative beta-pleated domain . These mutant proteins exhibit a markedly increased affinity for normal benzoate effectors, with K's values fivefold to 60-fold lower than those of the wild-type XylS protein . They are additionally activated by new effectors having certain substituents at position 2, 3 and 4 of the aromatic ring . Two other mutant proteins recognize new effectors having substituents at position 4 and 5 of the aromatic ring, and contain mutations at their C-terminal end within a putative alpha-helix-rich domain . Three other mutations, one of which leads to constitutive expression from Pm, each result in an amino acid change in the central region of the regulator . These findings suggest but do not prove that the effector binding pocket of the XylS protein may be composed of two or more non-contiguous segments of its primary structure . The XylS protein exhibits homology with the AraC protein of Escherichia coli, a protein that stimulates transcription from ara promoters when it is activated by arabinose or benzoate . Mutations influencing effector activation of the XylS protein characterized in this study are all located in regions exhibiting a high degree of homology with the corresponding aligned sequence of AraC protein.

J Biol Chem, 1990 Jan 15, 265(2), 660 - 5
Three isozymes of catechol 1,2-dioxygenase (pyrocatechase), alpha alpha, alpha beta, and beta beta, from Pseudomonas arvilla C-1; Nakai C et al.; Three isozymes of catechol 1,2-dioxygenase (pyrocatechase) from Pseudomonas arvilla C-1 were separated using DEAE-Toyopearl chromatography . The specific activities of each isozyme were similar to one another . The molecular weights of isozymes 1, 2, and 3 were estimated to be approximately 67,000, 64,000, and 59,000, respectively, from gel filtration . On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isozymes 1 and 3 gave a single protein band, corresponding to Mr = 32,000 and 30,000, respectively, and isozyme 2 gave two bands corresponding to Mr = 32,000 and 30,000 . These results indicated that isozymes 1 and 3 were homodimers, while isozyme 2 was a heterodimer . The NH2-terminal sequences up to 20 residues of these three isozymes confirmed that isozymes 1, 2, and 3 consisted of beta beta, alpha beta, and alpha alpha, respectively, based on our previous data (Nakai, C., Kagamiyama, H., Saeki, Y., and Nozaki, M . (1979) Arch . Biochem . Biophys . 195, 12-22) . Properties of these isozymes such as absorption spectrum, iron content, substrate specificity, and kinetic constants were similar to one another . Subunit exchange between the different isozymes and dissociation of the isozymes into subunits was not observed under nondenaturing conditions . Available evidence indicates that these isozymes exist naturally in the bacterium and were not due to artifacts caused by purification.

J Immunol, 1990 Jan 15, 144(2), 541 - 7
Requirements for the adoptive transfer of antibody responses to a priming antigen in man; Wimperis JZ et al.; Antibody responses to recall vaccines can be adoptively transferred after marrow transplantation in man . Transfer of responses to priming Ag has not been successful, although this would broaden the range of organisms to which recipients could be protected . To investigate the importance of T cells and Ag in such transfer we primed marrow donors with keyhole limpet hemocyanin (KLH) 1 or 3 wk before marrow harvesting . B cells secreting IgM and IgG anti-KLH antibody were present in donor marrow at both 1 and 3 wk after immunization . After T cell depletion, donor marrow was infused into chemo-irradiated recipients, half of whom were immunized pretransplant with KLH . We found no evidence for the transfer of the IgM component of the response . Clonal expansion of the transferred IgG antibody-secreting cells with a corresponding rise in recipient serum IgG antibody levels was seen only when donors were primed 3 wk before marrow harvest and when the recipients were also immunized . IEF and immunoblotting demonstrated that successful transfer coincided with maturation of the IgG primary response from a polyclonal to an oligoclonal pattern and confirmed that donor oligoclonal bands appeared in the recipient serum . We conclude that the immunization protocols required for the transfer of antibody responses to priming Ag reflect the initial dependence of unprimed B cells on T cell help and on prolonged Ag stimulation . Ag-stimulated primary B cells in T cell-depleted marrow respond only to the noncognate growth and differentiation signals available in the chemo-irradiated recipient after an initial period of clonal selection and expansion in the donor which is both T cell and Ag dependent . Even after this initial selection, continued expansion of antibody-secreting clones in recipients retains an absolute dependence on Ag stimulation . Immunization techniques to protect transplant recipients against organisms such as Pseudomonas and CMV may need to be modified accordingly.

Carbohydr Res, 1990 Jan 15, 195(2), 295 - 301
Structure of the putative O antigen containing 2-amino-2-deoxy-L-glucose in the reference strain for Pseudomonas cepacia serogroup O1; Cox AD et al.; Polymeric material isolated from the lipopolysaccharide of the reference strain of Pseudomonas cepacia serogroup O1 consisted mainly of D-glucose and 2-amino-2-deoxy-L-glucose: rhamnose and O-acetyl groups were also present . As a result of spectroscopic and degradative studies, the disaccharide repeating-unit shown could be assigned to the major polymer present . A possible origin of the minor components is suggested . ----4)-alpha-D-Glcp-(1----3)-alpha-L-GlcpNAc-(1----.

J Biol Chem, 1990 Jan 5, 265(1), 408 - 13
Interdomain hydrolysis of a truncated Pseudomonas exotoxin by the human immunodeficiency virus-1 protease; Tomasselli AG et al.; The specificity of HIV-1 (human immunodeficiency virus-1) protease has been evaluated relative to its ability to cleave the three-domain Pseudomonas exotoxin (PE66) and related proteins in which the first domain has been deleted or replaced by a segment of CD4 . Native PE66 is not hydrolyzed by the HIV-1 protease . However, removal of its first domain produces a molecule which is an excellent substrate for the enzyme . The major site of cleavage in this truncated exotoxin, called LysPE40, occurs in a segment that connects its two major domains, the translocation domain (II), and the ADP-ribosyltransferase (III) . This interdomain region contains the sequence ...Asn-Tyr-Pro-Thr.. . which is similar to that surrounding the scissile Tyr-Pro bond in the gag precursor polyprotein, a natural substrate of the HIV-1 protease . Nevertheless, it is not this sequence that is recognized and cleaved by the enzyme, but one 6 residues away, ...Ala-Leu-Leu-Glu.. . in which the Leu-Leu peptide bond is hydrolyzed . A second, slower cleavage takes place at the Leu-Ala bond 3 residues in from the NH2 terminus of LysPE40 . When domain I of PE66 is replaced by a segment comprising the first two domains of CD4, the resulting chimeric protein is hydrolyzed at the same Leu-Leu bond by HIV-1 protease . Enzyme activities toward synthetic peptides modeled after the sequences defined above in LysPE40 are in complete accord, relative to specificity, kinetics, and pH optimum, with results obtained in the hydrolysis of the parent protein . These findings demonstrate that ideas concerning the specificity of the HIV-1 protease that are based solely upon its processing of natural viral polyproteins can be expanded by evaluation of other multidomain proteins as substrates . Moreover, it would appear that it is not a particular conformation, but sequence and accessibility that play the dominant role in defining sites in a protein substrate that are susceptible to hydrolysis by the enzyme.

J Biol Chem, 1990 Jan 5, 265(1), 493 - 8
Tagetitoxin inhibits RNA synthesis directed by RNA polymerases from chloroplasts and Escherichia coli; Mathews DE et al.; Tagetitoxin, a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv . tagetis, inhibits RNA synthesis directed by chloroplast RNA polymerase . In isolated chloroplasts, tagetitoxin quickly and specifically reduced the incorporation of {3H}uridine into RNA . When it was added to transcriptionally active chloroplast protein extracts, the toxin directly inhibited incorporation of {32P}UTP into RNA . In addition, tagetitoxin inhibited in vitro RNA synthesis directed by the RNA polymerase from Escherichia coli . In vitro transcription reactions directed by chloroplast RNA polymerase or E . coli RNA polymerase are inhibited at tagetitoxin concentrations less than 1 microM . Nuclear RNA polymerase II purified from wheat germ was only affected at tagetitoxin concentrations greater than 100 microM during in vitro transcription . Tagetitoxin concentrations as high as 1 mM did not affect in vitro transcription reactions directed by RNA polymerase from bacteriophage T7 or SP6.

Biomed Biochim Acta, 1990, 49(4), 151 - 60
Interaction of bacterial glucose-6-phosphate dehydrogenase with triazine dyes: a study by means of affinity partitioning and kinetic analysis; Reuter R et al.; The interaction of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from Pseudomonas W6 with 33 triazine dyes bound to poly(ethylene glycol) has been screened by means of affinity partition in an aqueous two-phase system composed of poly-(ethylene glycol) and dextran . Regarding the strength of interaction expressed by the affinity partitioning effect three groups of dyes can be distinguished showing high, medium or low interaction to the enzyme . The different influence of the structurally related dyes Cibacronblue F3G-A, Procion Blue MX-3G and Procion Blue MX-R on the partitioning of glucose-6-phosphate dehydrogenase is discussed . Both coenzymes of this glucose-6-phosphate dehydrogenase, NADP+ and NAD+, are able to diminish the dye-enzyme interactions but to a different extent . In the case of Procion Red HE-3B less than 0.2 mM of NADP+ abolished completely the dye-protein interaction indicating that the dye binding site might be closely related to the nucleotide binding site . This does not hold for a series of other dyes revealing strong interactions to the enzyme . In these cases higher NADP(+)-concentrations are necessary to weaken the dye-enzyme interactions . Only small influences on the interaction between the dye and glucose-6-phosphate dehydrogenase were found with 1 mM NAD+ . The different effects of the two coenzymes are discussed with respect to separate binding sites for them and to different conformational states induced . The competition of Procion Red HE-3B with NADP+ and NAD+ for the binding site of glucose-6-phosphate dehydrogenase was also demonstrated by inhibition studies . The Ki-values of Procion Red HE-3B with NAD+ and NADP+, respectively, were calculated.

Minerva Urol Nefrol, 1990 Jan-Mar, 42(1), 39 - 42
{High-efficiency membranes and the dialysis solution}; Agliata S et al.; During this work through the total bacterial count and of the pseudomonas and the determination of the positive substances to Limulus Amebocyte Lysate test, bacterial pollution of the liquid bicarbonate concentrate has been determined together with the influence that preparation conditions and the entity of interval between the said preparation and utilization of concentrates have on it . Through the evaluation of the cardiac rate and the systemic arterial pressure, we have studied the influence of the bacterial pollution on the clinical of the patient . Analysing the obtained data we can point out that the liquid bicarbonate concentrate present an elevated degree of contamination which feels the effects of the production and storage procedures . The remark of inverse correlation between systemic arterial pressure and endotoxin level of the dialysate seems to reconfirm the importance of the bacterial contamination on the clinical of the patient.

Eur J Cardiothorac Surg, 1990, 4(6), 318 - 22
Lung and heart-lung transplantation for end-stage lung disease . The Bordeaux Lung and Heart-Lung Transplant Group; Couraud L et al.; Between February 1988 and December 1989, 15 combined heart-lung, 2 double lung and 5 single lung transplants were performed at our institution for end stage lung disease . The indication for heart-lung transplantation was primary lung disease with associated secondary heart failure in 11 cases, diffuse pulmonary disease with extensive adenopathy of the hilum in 2 cases and profuse and antibiotic-resistant tracheobronchial infection due to Pseudomonas in 2 cases . A double lung transplant was performed in 2 patients with hypertensive emphysema . The indication for a single lung transplantation was emphysema in 2 cases and pulmonary fibrosis in 3 cases; in this last indication, transplantation should be performed on the right side with a slight lengthening of the main bronchus to avoid the side-effects of mediastinal shift . There were 2 early deaths, 7 secondary deaths (from the 2nd to the 5th month) due to viral or bacterial infectious complications, and 1 late death in the 7th month (infection due to a syncitial virus) . All 12 surviving patients have an excellent functional result; the size of the tracheal or bronchial anastomosis ranges from 85% to 100% of normal . From this experience, we conclude that specificity and severity of lung hazards are mainly related to bronchial infection, dependence on steroids and pleural adhesions . Moreover, posttransplant pulmonary oedema, mucociliary dysfunction and the differential diagnosis between rejection and infection require careful endobronchial suction and periodical sampling.

Appl Biochem Biotechnol, 1990 Spring-Summer, 24-25, 171 - 82
Conversion of mannose to fructose by immobilized mannose isomerase from Pseudomonas cepacia; Allenza P et al.; The enzyme mannose isomerase (EC 5.3.1.7) catalyzes the isomerization of D-mannose and D-fructose . The conversion of mannose to fructose is the first step in the principal pathway of mannose dissimilation by Pseudomonas cepacia . This enzyme is induced during growth on medium containing mannose to levels three- to four-fold higher than observed during growth on glucose or citrate . Mannose isomerase was purified from extracts of mannose-grown P . cepacia and was efficiently immobilized onto a porous, noncompressible, ceramic support . The performance of the immobilized enzyme, compared with the soluble enzyme, was evaluated under a variety of operating conditions to examine its potential for use in a process for the production of fructose syrups containing a higher proportion of fructose than is currently possible using glucose isomerase.

Mikrobiol Zh, 1990 Jan-Feb, 52(1), 7 - 10
{The polysaccharide glycocalyx of Pseudomonas syringae pv . atrofaciens}; Romanenko VM; It was shown by the method of electron microscopy that cells of virulent strain Pseudomonas syringae rv . atrofaciens 4394 have extracellular, probably, polysaccharide glycocalix . It consists of acid components giving positive cytochemical reaction with ruthenium red, a specific reagent to polyanions.

Mol Gen Genet, 1990 Jan, 220(2), 294 - 300
Location and organization of the dimethylphenol catabolic genes of Pseudomonas CF600; Bartilson M et al.; The gene organization of the phenol catabolic pathway of Pseudomonas CF600 has been investigated . This strain can grow on phenol and some methylated phenols by virtue of an inducible phenol hydroxylase and metacleavage pathway enzymes . The genes coding for these enzymes are located on pVI150, an IncP-2 degradative mega plasmid of this strain . Twenty-three kilobases of contiguous DNA were isolated from lambda libraries constructed from strains harbouring wild type and Tn5 insertion mutants of pVI150 . A 19.9 kb region of this DNA has been identified which encodes all the catabolic genes of the pathway . Using transposon mutagenesis, polypeptide analysis and expression of subfragments of DNA, the genes encoding the first four enzymatic steps of the pathway have been individually mapped and found to lie adjacent to each other . The order of these genes is the same as that for isofunctional genes of TOL plasmid pWWO and plasmid NAH7.

Chemotherapy, 1990, 36(2), 117 - 26
Heterogeneity of beta-lactamase production in Pseudomonas maltophilia, a nosocomial pathogen; Cullmann W et al.; Twenty Pseudomonas maltophilia isolates were examined for susceptibility to beta-lactam antibiotics, including carbapenems, and for beta-lactamase production . All the isolates were resistant to imipenem (MICs 64-512 mg/l) and to a lesser extent, to meropenem (MICs 16-256 mg/l) . None of the isolates produced significant amounts of beta-lactamase without induction . Among the beta-lactams studied imipenem proved to be the most potent inducer; meropenem was a weaker inducer . Interestingly, 6-amino-penicillanic acid, even in concentrations up to 100 mg/l, entirely lacked induction activity . In any case enzyme production was drug concentration dependent and transient . Isoelectric focusing revealed 6 different enzymes distinguished by their different isoelectric points (pH 6.2, 8.3, 8.5, 9.0, 9.2 and 9.4) . This suggested the lack of a unique beta-lactamase profile in P . maltophilia . Addition of 5 mM cyclic AMP (cAMP) or 0.5 mM cAMP-N6, O2-dioctanoyl (a lipophilic derivative) resulted in a marked drop of beta-lactamase induction by imipenem as compared to the control assay . Monitoring of carbapenem hydrolysis by cell-free supernatants revealed inactivation of both carbapenems . Meropenem was inactivated about 5 times more rapidly than imipenem . Our studies revealed that beta-lactamase production in P . maltophilia as well as growth kinetics were influenced to a considerable extent by the nutrient medium employed.

J Clin Epidemiol, 1990, 43(2), 125 - 31
Effect of Pseudomonas cepacia colonization on survival and pulmonary function of cystic fibrosis patients; Lewin LO et al.; We conducted a historical prospective study of 124 cystic fibrosis (CF) patients colonized with Pseudomonas cepacia (cases) and 124 sex and age matched non-colonized CF patients (controls) . Thirty-two of the colonized patients died in the first year following P . cepacia colonization compared to 8 of the control patients, a highly significant difference (p less than 0.001) . In the second year, there was no significant difference in mortality between the two groups . Cases as a group had poorer pulmonary function and chest X-ray scores than controls up to 2 years before P . cepacia first appeared in their sputum or throat cultures . Regression analysis of pulmonary function tests (percent predicted FEV1 and RV/TLC) for each subject from 3 years before to 2 years after colonization revealed significant differences between cases and controls in slope for FEV1 and in slope and intercept for RV/TLC . When compared separately according to gender, the differences between cases and controls are significant in females but not in males . These results suggest that patients with poor pulmonary function are more prone to colonization with P . cepacia, that a subgroup of these patients will be dramatically affected and die within a year, and that the organism continues to exert a less dramatic negative effect on the pulmonary function of those patients who survive the initial acute effects of colonization, particularly in female patients.

J Clin Microbiol, 1990 Jan, 28(1), 143 - 5
Endocarditis associated with Comamonas acidovorans; Horowitz H et al.; A case of endocarditis caused by Comamonas acidovorans (Pseudomonas acidovorans) in a 42-year-old intravenous-drug abuser is described . This article appears to be the first detailed report of the isolation of this organism from a systemic clinical infection and its identification as a pathogen.

Neurosurgery, 1990 Jan, 26(1), 145 - 6
Intramedullary spinal cord abscess; Koppel BS et al.; Viral myelitis and bacterial epidural infections are common in intravenous drug abusers, but primary infections of the spinal cord are extremely rare . We report a 50-year-old active intravenous drug user who developed tetraplegia from an intramedullary abscess caused by Pseudomonas cepacia . Despite neurosurgical drainage and appropriate antibiotic therapy, no improvement was seen . Earlier intervention and a high index of suspicion is required in patients with a history of intravenous drug abuse and spinal cord symptoms.

J Bacteriol, 1990 Jan, 172(1), 477 - 80
Construction of a stable shuttle vector for high-frequency transformation in Pseudomonas syringae pv . syringae; Mukhopadhyay P et al.; A cryptic 80.3-kilobase plasmid, pOSU900, in Pseudomonas syringae pv . syringae strain J900 could be cured by treatment with mitomycin without affecting the pathogenicity of J900 on the host, Phaseolus vulgaris L . The replication region of pOSU900 was identified, subcloned, and modified for construction of a high-copy cloning vector . This vector could be transformed into Pseudomonas strains with high efficiency (ca . 10(6) transformants per microgram of DNA) and was very stable during growth of the host bacteria in planta.

J Bacteriol, 1990 Jan, 172(1), 465 - 8
Purification and properties of ferredoxinNAP, a component of naphthalene dioxygenase from Pseudomonas sp . strain NCIB 9816; Haigler BE et al.; One of the three components of the naphthalene dioxygenase occurring in induced cells of Pseudomonas sp . strain NCIB 9816 has been purified to homogeneity . The protein contained 2 g-atoms each of iron and acid-labile sulfur and had an apparent molecular weight of 13,600 . The evidence indicates that it is a ferredoxin-type protein that functions as an intermediate electron transfer protein in naphthalene dioxygenase activity.

J Bacteriol, 1990 Jan, 172(1), 457 - 64
Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp . strain NCIB 9816; Haigler BE et al.; Cells of Pseudomonas sp . strain NCIB 9816, after growth with naphthalene or salicylate, contain a multicomponent enzyme system that oxidizes naphthalene to cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene . We purified one of these components to homogeneity and found it to be an iron-sulfur flavoprotein that loses the flavin cofactor during purification . Dialysis against flavin adenine dinucleotide (FAD) showed that the enzyme bound 1 mol of FAD per mol of enzyme protein . The enzyme consisted of a single polypeptide with an apparent molecular weight of 36,300 . The purified protein contained 1.8 g-atoms of iron and 2.0 g-atoms of acid-labile sulfur and showed absorption maxima at 278, 340, 420, and 460 nm, with a broad shoulder at 540 nm . The purified enzyme catalyzed the reduction of cytochrome c, dichlorophenolindophenol, Nitro Blue Tetrazolium, and ferricyanide . These activities were enhanced in the presence of added FAD . The ability of the enzyme to catalyze the reduction of the ferredoxin involved in naphthalene reduction and other electron acceptors indicates that it functions as an NAD(P)H-oxidoreductase in the naphthalene dioxygenase system . The results suggest that naphthalene dioxygenase requires two proteins with three redox groups to transfer electrons from NADH to the terminal oxygenase.

Biomed Biochim Acta, 1990, 49(7), 539 - 46
Purification and characterization of glucose-6-phosphate dehydrogenase from Pseudomonas W6; Reuter R et al.; Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) has been purified from methanol grown Pseudomonas W6 by a simple procedure involving dye-ligand affinity chromatography on Cibacronblue F3G-A-Sephadex and Procion Red HE-3B-Sepharose . The purification procedure yielded a homogeneous enzyme with (1) high specific activity of 390 and 500 units/mg with NADP and NAD, respectively, and (2) low concentrations of contaminating activities . The molecular mass of the native enzyme was estimated to be 123 +/- 5 kDa . For the polypeptide chain after SDS denaturation a molecular mass of about 61 kDa was calculated . The kinetic behaviour of glucose-6-phosphate dehydrogenase exhibiting activity with either NADP or NAD was studied with respect to the substrate and coenzyme affinities and to ATP inhibition of enzyme activity . The applicability of prepared glucose-6-phosphate dehydrogenase as an auxiliary enzyme in clinical tests is discussed.

Zentralbl Mikrobiol, 1990, 145(6), 433 - 8
{Glyphosate utilization by Pseudomonas spec . GS}; Weidhase R et al.; Pseudomonas spec . GS grown in the presence of 14C-labelled glyphosate metabolizes the herbicide to inorganic phosphate as well as in respect of utilization of the C-skeleton . Furthermore it was shown that the primary step of degradation is the breakdown of the C-P-bond, because sarcosine was found as a transient metabolite.

Br J Neurosurg, 1990, 4(4), 279 - 85
Brain abscess: with special reference to infection by pseudomonas; Gupta SK et al.; Eighty cases of brain abscess treated in the University Hospital, BHU, Varanasi, India have been reviewed . Chronic suppurative otitis media was the commonest cause, followed by compound injuries . The overall mortality was 15% . In seven cases the causative organism was pseudomonas, resistant to most antibiotics . Prior to the availability of CT the mortality was 23.3%; after the routine use of CT for diagnosis the mortality fell to 10% . A high mortality (57%) was observed in patients who had pseudomonas . The best results were in patients who had been managed by excision of the abscess capsule.

Mikrobiologiia, 1990 Jan-Feb, 59(1), 156 - 62
{Storage of Pseudomonas strains degrading the surfactants}; Rotmistrov MN et al.; The survival rate and the destruction activity of Pseudomonas strains decomposing anionic and ampholytic surfactants were studied in the course of their storage for a long period of time . The strains were shown to remain viable and active after being freeze-dried for a year . Therefore, lyophilisation can be recommended as the main method of storage for bacterial strains decomposing the following surfactants: sulfoethoxylate, sulfonate, cyclimide, and amidobetaine.

Cornea, 1990, 9 Suppl 1, S41 - 3; discussion S47
Is your office safe? Yes; Cohen EJ; Contact lens (CL) fitting carries the risk of transmitting infectious agents, including adenovirus and Pseudomonas . Therefore, a number of precautions must be observed to ensure safety in the office . Paramount among these is hand washing, both immediately after contact with a patient's eyes and again between patients . Equally important is that all trial lenses and CLs removed from patients be disinfected before reuse . Low-water-content soft CLs can be heat disinfected; high-water-content CLs require chemical treatment . A combination of surfactant cleaning with a chlorhexidine-containing agent and hydrogen peroxide disinfection is preferred for rigid lenses to guarantee destruction of human immunodeficiency virus (HIV) . The proper use of lens care solutions is also necessary to minimize the risk of their becoming contaminated with pathogenic organisms . Only commercially prepared solutions should be used, preferably in small-volume bottles that are frequently replaced . Preservative-free solutions should be discarded after 1 day's use, whereas preserved solutions may be used for up to 2 weeks . Sterile saline rather than tap water is recommended for rinsing rigid lenses . Finally, part of the clinician's responsibility in running a safe office is to educate patients about these hygienic practices.

Cornea, 1990, 9 Suppl 1, S33 - 5; discussion S39-40
Acanthamoeba keratitis and contact lens wear: the patient is at fault; Moore MB; The avoidance of nonsterile solutions is important to curtailing Acanthamoeba keratitis, a serious infection that has been found to occur with all types of contact lens (CL) wear . Increased patient education, revised recommendations regarding the use of tap and distilled water, and improved disinfecting systems are vital to preventing infection . These precautions are particularly important since it appears that Acanthamoeba, unlike Pseudomonas, may not require an epithelial defect to establish corneal infection and, once in the cornea, may not respond to drug therapy or surgical extirpation . Unfortunately, many patients receive poor lens care instructions or cannot be relied on to follow appropriate routines . Finding a foolproof means of lens disinfection for them is critical . Recently, several disinfection systems were tested against Acanthamoeba castellanii and Acanthamoeba polyphaga cysts and trophozoites to see which might prove most effective . Effective systems included heat disinfection at 70 to 80 degrees C for 10 min, 3% hydrogen peroxide for 2-3 h, 0.001% thimerosal with edetate for 4 h, 0.005% benzalkonium chloride with edetate and reagent for 4 h, and either 0.001% chlorhexidine for 4 h or 0.004% chlorhexidine for 1 h . Thus, for patients who are careless or persist in using nonsterile rinsing solutions, it appears that at least some methods of disinfection will help prevent Acanthamoeba infection.

Can J Microbiol, 1990 Jan, 36(1), 56 - 9
Determination of peptidyl dipeptidase activity in 24 bacterial species; Stevens J et al.; Of 24 bacterial species examined for lisinopril refractive peptidyl dipeptidase activity, only 8 contained activity . Activity in Pseudomonas maltophilia was more than fourfold higher than that of any other species . Pseudomonas maltophilia may be unique among bacteria in possessing high peptidyl dipeptidase activity that is both EDTA inhibitable and lisinopril resistant.

Arch Microbiol, 1990, 154(4), 407 - 9
Regulation of pyrimidine biosynthesis in Pseudomonas cepacia; West TP et al.; Pyrimidine biosynthesis was investigated in Pseudomonas cepacia ATCC 17759 . The presence of the de novo pyrimidine biosynthetic pathway enzyme activities was confirmed in this strain . Following transposon mutagenesis of the wild-type cells, a mutant strain deficient for orotidine 5'-monophosphate decarboxylase activity (pyrF) was isolated . Uracil, cytosine or uridine supported the growth of this mutant . Uracil addition to minimal medium cultures of the wild-type strain diminished the levels of the de novo pyrimidine biosynthetic enzyme activities, while pyrimidine limitation of the mutant cells increased those de novo enzyme activities measured . It was concluded that regulation of pyrimidine biosynthesis at the level of enzyme synthesis in P . cepacia was present . Aspartate transcarbamoylase activity was found to be regulated in the wild-type cells . Its activity was shown to be controlled in vitro by inorganic pyrophosphate, adenosine 5'-triphosphate and uridine 5'-phosphate.

Biochem Int, 1990, 20(3), 591 - 7
Enzyme interactions of 2,10-ethanoandrostene-3,17-dione: aromatase and 17 beta-hydroxysteroid dehydrogenase; Greway AT et al.; An analogue of androstenedione containing an ethano bridge between carbons 2 and 10 of the A ring of the steroid, 1, has been evaluated as an inhibitor and a possible substrate of human placental aromatase . This compound was found to be a competitive inhibitor versus androstenedione (Kis = 25 +/- 2 nM) of the aromatase activity . Analyses of the incubation mixtures of 1 with human placental microsomes and NADPH by GC-MS indicated the formation of a new compound having an increase in molecular weight of 2 mass units (300 m.u.) from that of the parent steroid (298 m.u.) . Subsequent analyses of incubations of 1 with an isolated 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) from Pseudomonas testosteronii in the presence of NADPH resulted in the formation of a new compound having the same retention time and molecular mass as that found for the product from the placental microsome incubation . Consequently, steroid 1 is both an inhibitor of human placental aromatase and a substrate for 17 beta-HSD.

Mol Gen Genet, 1990 Jan, 220(2), 222 - 8
Repeated sequences including RS1100 from Pseudomonas cepacia AC1100 function as IS elements; Haugland RA et al.; Several lines of evidence were obtained that the previously identified, repeated sequence RS1100 of Pseudomonas cepacia strain AC1100 undergoes transposition events . DNA sequences flanking the chlorohydroxy hydroquinone (CHQ) degradative genes of this organism were examined from sources, including several independently isolated cosmid clones from an AC1100 genomic library and genomic DNAs of two independently maintained wild-type AC1100 isolates . Hybridization and restriction endonuclease mapping studies revealed these sequences to be similar except for their numbers and distributions of RS1100 copies . A recombinant plasmid containing the immediate chq gene region and excluding any copies of RS1100 was conjugated into AC1100 mutant RHA5 which was shown to have undergone a deletion of its corresponding DNA . Hybridization and restriction mapping analyses of several reisolated plasmids revealed the presence of RS1100 sequences at different positions within either the vector or insert portions . One such plasmid contained tandem copies of RS1100 with an intervening DNA sequence also of AC1100 origin . Similar experiments involving introduction of the promoter probe plasmid pKT240 into wild-type AC1100 cells resulted in the acquisition of high-concentration streptomycin resistance by a number of recipients . The reisolated plasmids in most cases also conferred streptomycin resistance to Escherichia coli transformants and in each case were found to contain insertions close to the upstream portion of the aphC structural gene . These insertions alternatively contained RS1100 sequences for a newly identified 3400 bp repeated sequence from AC1100 . Based on these results, RS1100 has been redesignated as insertion sequence IS931 and the 3400 bp repeated sequence has been designated as IS932.

Proc Natl Acad Sci U S A, 1990 Jan, 87(1), 308 - 12
Pseudomonas exotoxin contains a specific sequence at the carboxyl terminus that is required for cytotoxicity; Chaudhary VK et al.; Pseudomonas exotoxin (PE), a single-chain polypeptide toxin of 613 amino acids, consists of three functional domains: an amino-terminal receptor-binding domain, a middle translocation domain, and a carboxyl-terminal ADP-ribosylation domain . Deletion of as few as 2 or as many as 11 amino acids from the carboxyl terminus of PE does not affect ADP-ribosylation activity but produces noncytotoxic molecules . Deletions and substitutions between positions 602 and 611 of PE show that the last 5 amino acids of PE are very important for its cytotoxic action . The carboxyl-terminal sequence of PE is Arg-Glu-Asp-Leu-Lys . Mutational analysis indicates that a basic amino acid at 609, acidic amino acids at 610 and 611, and a leucine at 612 are required for full cytotoxic activity . Lysine at 613 can be deleted or replaced with arginine but not with several other amino acids . Mutant toxins are able to bind normally to target Swiss mouse 3T3 cells and are internalized by endocytosis, but apparently they do not penetrate into the cytosol . A PE molecule that ends with Lys-Asp-Glu-Leu, which is a well defined endoplasmic reticulum retention sequence {Munro, S . and Pelham, R . B . (1987) Cell 48, 899-907}, is fully cytotoxic, suggesting that a common factor may be involved in intoxication of cells by PE and retention of proteins in the lumen of the endoplasmic reticulum . Sequences similar to those at the carboxyl end of PE are also found at the end of Cholera toxin A chain and Escherichia coli heat-labile toxin A chain.

J Med Genet, 1990 Jan, 27(1), 17 - 20
The genotype of a new linked DNA marker, MP6d-9, is related to the clinical course of cystic fibrosis; Gasparini P et al.; The clinical symptoms of a cohort of cystic fibrosis patients were related to their genotypes using RFLPs shown with MspI and the closely linked DNA marker MP6d-9 . In the majority of CF chromosomes, the restriction site for MspI was present, and the genotype 2/2 was found most often in patients who were severely affected by the disease . The genotype 1/2 was significantly over-represented in patients with very mild clinical manifestations, including pancreatic sufficiency, absence of meconium ileus, and absence of Pseudomonas colonisation . When pancreatic dysfunction was present, the 1/2 genotype was associated with a mild form, while the 2/2 genotype was found in patients with severe insufficiency . None of our patients had the 1/1 genotype . These results indicate that the newly isolated MP6d-9 marker correlates with some important symptoms of cystic fibrosis.

Bioorg Khim, 1990 Jan, 16(1), 90 - 7
{Antigenic polysaccharides of bacteria . 37 . Structure of the polysaccharide chain of Pseudomonas syringae pv.tabaci (serogroup VII) lipopolysaccharide}; Shashkov AS et al.; The structure of the O-specific polysaccharide chain of Pseudomonas syringae pv . tabaci strain 223 (serogroup VII) lipopolysaccharide was established on the basis of one- and two-dimensional 1H NMR analysis, 13C NMR analysis and calculation of optical rotation . The structure determined by the non-destructive way was confirmed by acid hydrolysis and methylation . (Sequence: see text) . O-Antigen of the strain studied is similar in structure and serological properties to O-antigens of Pseudomonas syringae strains belonging to serogroup I.

Biodegradation, 1990, 1(1), 43 - 53
Phosphonate utilization by bacteria in the presence of alternative phosphorus sources; Schowanek D et al.; Batch and continuous culture experiments were carried out to investigate the effect of orthophosphate and p-nitrophenylphosphate on the utilization of various phosphonates as a P source by bacteria . Detailed tests with methylphosphonate as a model phosphonate and the phosphonate-degrading Pseudomonas paucimobilis strain MMM101a revealed that, in contrast with the majority of literature data, the phosphates did not suppress phosphonate utilization . Under conditions of P stress, strain MMM101a simultaneously took up both P-sources, with a preference for the phosphate-P . Study of the kinetic parameters for strain MMM101a, growing on the different P sources revealed similar, rather low, maximum growth rates (ca . 0.15 h-1) . However, the affinity for orthophosphate (Ks:0.17 microM), was more than two orders of magnitude higher than for methylphosphonate (Ks: 66 microM), which might account for the preferential uptake of orthophosphate . Cellular phosphorus yields in continuous cultures varied considerably with the conditions applied . The results suggest that phosphonate degradation can occur also in environments with substantial backgrounds of phosphate.

Phytochemistry, 1990, 29(8), 2411 - 4
Characterization of maize polyamine oxidase; Federico R et al.; Some structural and biochemical characteristics of polyamine oxidase (PAO) purified from maize shoots have been examined . The enzyme has only alanine as N-terminal amino acid and its N-terminal sequence shows a significant degree of homology with tryptophan 2-monooxygenase from Pseudomonas syringae pv . savastanoi . The pH optimum for the stability of the native enzyme is 5, similar to that of the barley leaf enzyme . Calorimetric analysis shows a single two-state transition at pH 6 with Tm 49.8 degrees . At pH 5 the thermal stability is increased by more than 14 degrees . Amine oxidation products, delta 1-pyrroline and diazabicyclononane, are competitive inhibitors of PAO activity (apparent Ki = 400 and 100 microM respectively) . Moreover these compounds improve the thermal stability of the enzyme . N1-Acetylspermine, which is a good substrate for mammalian PAO, acts as a non-competitive inhibitor for the plant enzyme.

Carbohydr Res, 1989 Dec 21, 195(1), 123 - 9
Structures of the O-specific polymers from the lipopolysaccharides of the reference strains for Pseudomonas cepacia serogroups O3 and O5; Cox AD et al.; The putative O-specific polymers of lipopolysaccharides from two reference strains of Pseudomonas cepacia have been isolated and characterized . Both polymers have disaccharide repeating-units . Structure 1 was established for the O3 polymer, and structure 2 for the O5 polymer . Polymers with the same repeating units have been found previously as the O antigens of other bacteria . ----2)-beta-D-Ribf-(1----4)-alpha-D-GalpNAc-(1---- ----4)-alpha-L-Rhap-(1----3)-beta-D-ManpNAc-(1----

Gene, 1989 Dec 21, 85(1), 233 - 8
Nucleotide sequence and expression of the catechol 2,3-dioxygenase-encoding gene of phenol-catabolizing Pseudomonas CF600; Bartilson M et al.; Pseudomonas CF600 degrades phenol and some of its methylated derivatives via a plasmid-encoded catabolic pathway . The catechol 2,3-dioxygenase (C23O) enzyme of this pathway catalyses the conversion of catechol to 2-hydroxymuconic semialdehyde . We have determined the nucleotide (nt) sequence of the dmpB structural gene for this enzyme, and expressed and identified its polypeptide product in Escherichia coli . The xylE gene of TOL plasmid pWWO and the nahH gene of plasmid NAH7 encode analogous C23O enzymes . Comparison of these three genes shows homology of 78-81% on the nt level and 83-87% homology on the amino acid level.

J Biol Chem, 1989 Dec 5, 264(34), 20467 - 73
Spectroscopic characterization of secondary amine mono-oxygenase . Comparison to cytochrome P-450 and myoglobin; Alberta JA et al.; Secondary amine mono-oxygenase from Pseudomonas aminovorans catalyzes the NAD(P)H- and dioxygen-dependent N-dealkylation of secondary amines to yield a primary amine and an aldehyde . Heme iron, flavin, and non-heme iron prosthetic groups are known to be present in the oligomeric enzyme . The N-dealkylation reaction is also catalyzed by the only other heme-containing mono-oxygenase, cytochrome P-450 . In order to identify the heme iron axial ligands of secondary amine mono-oxygenase so as to better define the structural requirements for oxygen activation by heme enzymes, we have investigated the spectroscopic properties of the enzyme . The application of three different spectroscopic techniques, UV-visible absorption, magnetic circular dichroism and electron paramagnetic resonance, to study eight separate enzyme derivatives has provided extensive and convincing evidence for the presence of a proximal histidine ligand . This conclusion is based primarily on comparisons of the spectral properties of the enzyme with those of parallel derivatives of myoglobin (histidine proximal ligand) and P-450 (cysteinate proximal ligand) . Spectral studies of ferric secondary amine mono-oxygenase as a function of pH have led to the proposal that the distal ligand is water . Deprotonation of the distal water ligand occurs upon either raising the pH to 9.0 or substrate (dimethylamine) binding . In contrast, the deoxyferrous enzyme appears to have a weakly bound nitrogen donor distal ligand . Initial spectroscopic studies of the iron-sulfur units in the enzyme are interpreted in terms of a pair of Fe2S2 clusters . Secondary amine mono-oxygenase is unique in its ability to function as cytochrome P-450 in activating molecular oxygen but to do so with a myoglobin-like active site . As such, it provides an important system with which to probe structure-function relations in heme-containing oxygenases.

J Clin Microbiol, 1989 Dec, 27(12), 2640 - 6
Subgrouping of Pseudomonas cepacia by cellular fatty acid composition; Mukwaya GM et al.; The cellular fatty acid compositions were determined for 42 strains of Pseudomonas cepacia from five cystic fibrosis centers in North America . All isolates contained significant (20%) amounts of hexadecanoic (C16:0), and cis-9 hexadecenoic (C16:1 cis9) acids and an isomer of octadecenoic acid (C18:1) . None had hydroxy acids containing fewer than 14 carbon atoms . The quantitative data from the fatty acid analysis were highly reproducible and provided a basis for numerical analysis . Five subgroups comprising all the strains were obtained by cluster analysis and further characterized by principal-component analysis . With minor exceptions, the predominant subgroup identified in each center was different from that identified in other centers and accounted for one-half of the isolates within each center . Cellular fatty acid composition is a useful adjunct to biochemical characterization for the identification of P . cepacia isolated from cystic fibrosis patients . Numerical analysis of the fatty acid data can separate P . cepacia into subgroups, which may provide useful epidemiologic information or a basis for further analysis by more complex techniques such as DNA probe analysis.

J Bacteriol, 1989 Dec, 171(12), 6791 - 9
Cloning of 1,2-dichloroethane degradation genes of Xanthobacter autotrophicus GJ10 and expression and sequencing of the dhlA gene; Janssen DB et al.; A gene bank from the chlorinated hydrocarbon-degrading bacterium Xanthobacter autotrophicus GJ10 was prepared in the broad-host-range cosmid vector pLAFR1 . By using mutants impaired in dichloroethane utilization and strains lacking dehalogenase activities, several genes involved in 1,2-dichloroethane metabolism were isolated . The haloalkane dehalogenase gene dhlA was subcloned, and it was efficiently expressed from its own constitutive promoter in strains of a Pseudomonas sp., Escherichia coli, and a Xanthobacter sp . at levels up to 30% of the total soluble cellular protein . A 3-kilobase-pair BamHI DNA fragment on which the dhlA gene is localized was sequenced . The haloalkane dehalogenase gene was identified by the known N-terminal amino acid sequence of its product and found to encode a 310-amino-acid protein of molecular weight 35,143 . Upstream of the dehalogenase gene, a good ribosome-binding site and two consensus E . coli promoter sequences were present.

J Bacteriol, 1989 Dec, 171(12), 6782 - 90
Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway; Abril MA et al.; The TOL plasmid upper pathway operon encodes enzymes involved in the catabolism of aromatic hydrocarbons such as toluene and xylenes . The regulator of the gene pathway, the XylR protein, exhibits a very broad effector specificity, being able to recognize as effectors not only pathway substrates but also a wide variety of mono- and disubstituted methyl-, ethyl-, and chlorotoluenes, benzyl alcohols, and p-chlorobenzaldehyde . Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, two upper pathway enzymes, exhibit very broad substrate specificities and transform unsubstituted substrates and m- and p-methyl-, m- and p-ethyl-, and m- and p-chloro-substituted benzyl alcohols and benzaldehydes, respectively, at a high rate . In contrast, toluene oxidase only oxidizes toluene, m- and p-xylene, m-ethyltoluene, and 1,2,4-trimethylbenzene {corrected}, also at a high rate . A biological test showed that toluene oxidase attacks m- and p-chlorotoluene, albeit at a low rate . No evidence for the transformation of p-ethyltoluene by toluene oxidase has been found . Hence, toluene oxidase acts as the bottleneck step for the catabolism of p-ethyl- and m- and p-chlorotoluene through the TOL upper pathway . A mutant toluene oxidase able to transform p-ethyltoluene was isolated, and a mutant strain capable of fully degrading p-ethyltoluene was constructed with a modified TOL plasmid meta-cleavage pathway able to mineralize p-ethylbenzoate . By transfer of a TOL plasmid into Pseudomonas sp . strain B13, a clone able to slowly degrade m-chlorotoluene was also obtained.

J Bacteriol, 1989 Dec, 171(12), 6468 - 72
Nucleotide sequence and expression in Escherichia coli of the 3-hydroxy-3-methylglutaryl coenzyme A lyase gene of Pseudomonas mevalonii; Anderson DH et al.; The mva operon of Pseudomonas mevalonii encodes two enzymes that can convert internalized mevalonate into acetoacetate and acetyl-coenzyme A (CoA) . The promoter-proximal gene of this operon is mvaA, the structural gene for 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (EC 1.1.1.88) . The cloning, characterization, and expression of mvaA has been reported (M . J . Beach and V . W . Rodwell, J . Bacteriol . 171:2994-3001, 1989) . We report here the nucleotide sequence of another gene of this operon, mvaB, its expression in Escherichia coli, and its identification as the structural gene for HMG-CoA lyase (EC 4.1.3.4) . P . mevalonii HMG-CoA lyase is a cytosolic protein with 301 amino acid residues and a molecular weight of 31,600 . This represents the first reported sequence of an HMG-CoA lyase from any source.

J Clin Oncol, 1989 Dec, 7(12), 1932 - 42
Immunotoxins: a clinical review of their use in the treatment of malignancies; Hertler AA et al.; Immunotoxins are a new class of antitumor agents consisting of tumor-selective ligands (generally monoclonal antibodies {MoAbs}) linked to highly toxic protein molecules that have been modified to remove their normal tissue-binding domains . These immuno-conjugates combine the potency of the parent toxin with the specificity of the attached ligand . Toxins used in the construction of immunotoxins belong to a group of peptides that catalytically inhibit the elongation step of protein synthesis, and include ricin, abrin, pokeweed antiviral protein, gelonin, Pseudomonas exotoxin A, diptheria toxin, and alpha-sarcin . To synthesize immunotoxins, the normal cell-binding function must be removed by chemical cleavage or modification, or in the case of toxins that have been cloned, genetic engineering used to delete amino acids critical to cell binding . Covalent linkage of toxin to ligand generally involves a disulfide or thioether bond, though recently, recombinant toxin molecules with ligands that are genetically engineered into the protein have been made . The most successful clinical application of immunotoxins has been in the depletion of T cells from allogeneic bone marrow grafts to prevent graft-versus-host disease (GVHD) . Clinical trials have been conducted using immunotoxins for the systemic treatment of chronic lymphocytic leukemia (CLL), GVHD, and selected solid tumors . With the possible exception of GVHD, responses have been limited . Obstacles have included rapid systemic clearance, poor delivery to extravascular tumor deposits, and humoral immune responses to the immunotoxin . Research to overcome these problems is in progress and should lead to a better definition of the role of immunotoxins in the therapy of malignancies.

Indian J Biochem Biophys, 1989 Dec, 26(6), 410 - 2
Preliminary studies on excretory components of a virulent strain (F-2) of Pseudomonas solanacearum; Basu S et al.; The excretory material (EM) was isolated from the culture medium of a virulent strain (F-2) of Pseudomonas solanacearum . Electrophoretic studies depicted the heterogeneous nature of EM and the presence of released lipopolysaccharide in it . Both the exopolysaccharide and the released LPS contained D-galactose as the major sugar constituent together with D-glucose, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine . In addition, L-rhamnose was present as a constituent sugar of the released LPS.

FASEB J, 1989 Dec, 3(14), 2647 - 52
Cytotoxic activities of a fusion protein comprised of TGF alpha and Pseudomonas exotoxin; Siegall CB et al.; A cDNA encoding transforming growth factor type alpha (TGF alpha) was fused to the 5' end of a gene encoding a modified form of Pseudomonas exotoxin A (PE), which is devoid of the cell recognition domain (domain Ia) . The chimeric molecule, termed TGF alpha-PE40, was expressed in Escherichia coli and isolated from the periplasm or inclusion bodies depending on the construction expressed . TGF alpha-PE40 was found to be extremely cytotoxic to cells displaying epidermal growth factor (EGF) receptors . Comparison with a similar molecule in which TGF alpha was placed at the carboxyl end of PE40 demonstrated the importance of the position of the cell recognition element; TGF alpha-PE40 was found to be about 30-fold more cytotoxic to cells bearing EGF receptors than PE40-TGF alpha . In addition, TGF alpha-PE40 was shown to be extremely cytotoxic to a variety of cancer cell lines including liver, ovarian, and colon cancer cell lines, indicating high levels of EGF receptor expression in these cells.

J Immunol, 1989 Dec 1, 143(11), 3498 - 502
Selective immunosuppression of activated T cells with the chimeric toxin IL-2-PE40 . Inhibition of experimental autoimmune uveoretinitis; Roberge FG et al.; A characteristic of activated T lymphocytes is the expression of high affinity IL-2R . We studied a new method of selective immunosuppression directed against activated T cells by using a chimeric recombinant protein (IL-2-PE40) composed of IL-2 fused to a modified Pseudomonas exotoxin lacking its cell recognition domain . As a model of T cell-mediated disease, we used experimental autoimmune uveoretinitis (EAU) produced in Lewis rats by active immunization with the retinal S-Ag . The treatment protocol consisted of i.p . injection of IL-2-PE40 at 0.25 micrograms/g every 12 h . Controls were PBS, PE40, or IL-2-PE40asp553 a mutant form of the molecule with reduced activity . Treatment with IL-2-PE40 resulted in a significant reduction of the incidence and severity of EAU over controls . The analysis of the effect of i.p . injection of IL-2-PE40 on the popliteal draining lymph nodes of immunized animals showed a marked reduction in the lymphocytes content . Transfer experiments demonstrated that IL-2-PE40 prevented the development of EAU effector T cells . Interestingly, although activated B cells were reported to express IL-2R, there was no significant reduction of antibody production against the immunizing Ag under IL-2-PE40 treatment, suggesting sparing of the B cells.

Proc Natl Acad Sci U S A, 1989 Dec, 86(23), 9539 - 43
CD4-Pseudomonas exotoxin hybrid protein blocks the spread of human immunodeficiency virus infection in vitro and is active against cells expressing the envelope glycoproteins from diverse primate immunodeficiency retroviruses; Berger EA et al.; We previously described an unusual recombinant protein, designated CD4(178)-PE40, containing the gp120 binding region of human CD4 linked to active regions of Pseudomonas exotoxin A . The ability of this molecule to selectively inhibit protein synthesis in cells expressing the surface envelope glycoprotein of human immunodeficiency virus (HIV) suggested this molecule may be useful in treating infected individuals . To further evaluate its therapeutic potential, several in vitro properties of this hybrid toxin were examined . CD4(178)-PE40 was found to be an extremely potent cytotoxic agent, selectively killing HIV-infected cells with IC50 values around 100 pM . In a coculture system employing mixtures of HIV-infected and -uninfected cells, the hybrid toxin inhibited spread of the infection, as judged by a delay in HIV-induced cell killing and a dramatic suppression of free virus production . Experiments with control recombinant proteins indicated that this protective effect was primarily due to selective killing of the HIV-infected cells, rather than to a simple blocking effect of the CD4 moiety of the hybrid toxin . Using recombinant vaccinia viruses as expression vectors, we found the hybrid toxin to be active against cells expressing the envelope glycoproteins of divergent isolates of HIV-1, as well as HIV-2 and simian immunodeficiency virus . These results provide further support for the therapeutic potential of CD4(178)-PE40 in the treatment of HIV-infected individuals.

J Biol Chem, 1989 Nov 25, 264(33), 19659 - 65
Purification and properties of the phosphotriesterase from Pseudomonas diminuta; Dumas DP et al.; The phosphotriesterase produced from the opd cistron of Pseudomonas diminuta was purified 1500-fold to homogeneity using a combination of gel filtration, ion exchange, hydrophobic, and dye matrix chromatographic steps . This is the first organophosphate triesterase or organophosphofluoridate hydrolyzing enzyme to be purified to homogeneity . The enzyme is a monomeric, spherical protein having a molecular weight of 39,000 . A single zinc atom is bound to the enzyme and is required for catalytic activity . Incubation with metal chelating compounds, o-phenanthroline, EDTA, or 2,6-pyridine dicarboxylate inactivate the enzyme . The kinetic rate constants, kcat and kcat/Km, for the hydrolysis of paraoxon are 2100 s-1 and 4 x 10(7) M-1 s-1, respectively . The enzyme is inhibited competitively by dithiothreitol, dithioerithritol, and beta-mercaptoethanol . In addition to paraoxon the phosphotriesterase was found to hydrolyze the commonly used organophosphorus insecticides, dursban, parathion, coumaphos, diazinon, fensulfothion, methyl parathion, and cyanophos.

J Biol Chem, 1989 Nov 5, 264(31), 18818 - 23
Identification of the carboxyl-terminal amino acids important for the ADP-ribosylation activity of Pseudomonas exotoxin A; Chow JT et al.; The ADP-ribosylation domain of Pseudomonas exotoxin A (PE) has been identified to reside in structural domain III (residues 405-613) and a portion of domain Ib (residues 385-404) of the molecule (Hwang, J., FitzGerald, D . J., Adhya, S., and Pastan, I . (1987) Cell 48, 129-136) . To further determine the carboxyl end region essential for ADP-ribosylation activity, we constructed sequential deletions at the carboxyl-terminal of PE . Our results show that a clone with a deletion of the carboxyl-terminal amino acid residues from Arg-609 to Lys-613 and replaced with Arg-Asn retained wild-type PE ADP-ribosylation activity . Deletion of the terminal amino acid residues from Ala-596 to Lys-613 and replaced with Val-Ile-Asn reduced ADP-ribosylation activity by 75%, while deletions of 36 or more amino acids from the carboxyl terminus completely lose their ADP-ribosylation activity . These modified PEs were also examined for their ability to block PE cytotoxicity . Our results shown that modified PEs which lost their ADP-ribosylation activity correspondingly lost their cytotoxicity . Furthermore, extracts containing PE fragments without ADP-ribosylation activity were able to block the cytotoxic activity of intact PE . Our results thus indicate that carboxyl-terminal amino acids in the Ser-595 region are crucial for ADP-ribosylation activity and, consequently, cytotoxicity of PE . The modified PEs which have lost their ADP-ribosylation activity may also be a route to new PE vaccines.

J Bacteriol, 1989 Nov, 171(11), 6294 - 9
Removal of CO dehydrogenase from Pseudomonas carboxydovorans cytoplasmic membranes, rebinding of CO dehydrogenase to depleted membranes, and restoration of respiratory activities; Jacobitz S et al.; In Pseudomonas carboxydovorans, CO dehydrogenase and hydrogenase were found in association with the cytoplasmic membrane in a weakly bound and a tightly bound pool . The pools could be experimentally distinguished on the basis of resistance to removal by washes in low-ionic-strength buffer . The tightly bound pool of the enzymes could be differentially solubilized under conditions leaving the electron transport system intact and with the nondenaturing zwitterionic detergent 3-(3-cholamidopropyl) dimethylammonio 1-propane-sulfonic acid (CHAPS) and the nonionic detergent dodecyl beta-D-maltoside . In vitro reconstitution of depleted membranes with the corresponding supernatants containing CO dehydrogenase led to binding of the enzyme and to reactivation of respiratory activities with CO . The reconstitution reaction required cations with effectiveness which increased with increasing ionic charge: monovalent (Li+), divalent (Mg2+, Mn2+), or trivalent (Cr3+, La3+) . Reconstitution of depleted membranes with CO dehydrogenase was specific for CO-grown bacteria . Cytoplasmic membranes from H2- or heterotrophically grown Pseudomonas carboxydovorans had no affinity for CO dehydrogenase at all, indicating the absence of the physiological electron acceptor of the enzyme, which presumably is cytochrome b561, or another membrane anchor.

Cell Calcium, 1989 Nov-Dec, 10(8), 511 - 7
A calcium flux at the termination of replication triggers cell division in Escherichia coli . Hypothesis; Norris V; Cell division in Escherichia coli is coupled to chromosome replication . Even in the absence of known inducible division inhibitors, perturbations of chromosome replication affect cell division . Early studies suggested that a signal at the termination of replication might trigger subsequent division . Although later studies have suggested that fork encounter during termination is an active process involving specific termination sites and the tus protein, the coupling mechanism between termination and cell division remains to be elucidated . Recently it has been shown that the chromosome of a bacterium, Pseudomonas tabaci, contains a high proportion of calcium . E . coli maintains an intracellular concentration of free calcium identical to that of higher organisms and in dividing cells of E . coli a twenty-fold increase in the level of total calcium in the cytoplasm, a flux, occurs . In this article I propose that during the replication of the chromosome calcium entry balances calcium binding to DNA . At the termination of replication, there is a brief interval between the end of calcium binding to the chromosome and the end of calcium entry or release into the cytoplasm . During this interval the level of free calcium therefore rises . This rise may result in the observed flux by triggering the entry of calcium directly via voltage-gated calcium channels or indirectly via changes in phospholipid configurations . Mechanisms whereby these changes in calcium levels might be coupled to cell division and to a phospholipid control of the cell cycle are discussed.

Am J Kidney Dis, 1989 Nov, 14(5 Suppl 2), 45 - 53
The multichain interleukin-2 receptor: a target for immunotherapy of patients receiving allografts; Waldmann TA et al.; Antigen-induced activation of resting T cells induces the synthesis of interleukin-2 (IL-2) as well as the expression of specific cell surface high-affinity receptors for this lymphokine . There are at least two forms of the cellular receptors for IL-2, one with a very high affinity and the other with a lower affinity . Two IL-2 binding peptides, a 55-kd peptide reactive with the anti-Tac monoclonal antibody and a 75-kd, non-Tac IL-2-binding peptide, were identified . A multichain model for the high-affinity receptor in which an independently existing p55 or p75 peptide would represent low-or intermediate-affinity receptors, respectively, and high-affinity receptors would be expressed when both of these receptors are expressed and associated in a receptor complex, is proposed . An additional 95 - to 105-kd peptide may also participate in the multisubunit, high-affinity form of the IL-2 receptor . The p75 peptide is receptor for IL-2 on large granular lymphocytes and is sufficient for the IL-2 activation of these cells . In contrast to resting T cells, the T cells of patients with certain neoplasias of mononuclear cells and of patients with select autoimmune disorders, as well as T cells participating in organ allograft rejections, express the Tac antigen . To exploit the fact that IL-2 receptors are present on abnormally activated T cells but no on normal resting T cells, clinical trials have been initiated involving patients with neoplastic or autoimmune disorders as well as those receiving organ allografts . These patients are being treated with unmodified anti-Tac, with isotopic (212 Bi and 90Y) chelates of anti-Tac, with truncated Pseudomonas toxin conjugates of anti-Tac or IL-2, and with recombinant chimeric "humanized" anti-Tac.

J Bacteriol, 1989 Nov, 171(11), 6218 - 26
Molecular cloning and sequence analysis of the structural gene of ferredoxin I from the photosynthetic bacterium Rhodobacter capsulatus; Schatt E et al.; The structural gene (fdxN) encoding ferredoxin I (FdI) in the photosynthetic bacterium Rhodobacter capsulatus was isolated from a cosmid library by using an oligonucleotide probe corresponding to the N-terminal amino acid sequence of FdI . The nucleotide sequences of the gene and of the 3'- and 5'-flanking regions were determined . The gene fdxN codes for a polypeptide of 64 mino acids having a calculated molecular weight of 6,728 . Amino acid sequencing of the N- and C-terminal ends of FdI allowed the determination of 86% of the primary structure and confirmed that FdI is the fdxN gene product . Sequence comparisons indicate that FdI shares common structural features with ferredoxins containing two {4Fe-4S} clusters, including eight conserved cysteines . Maximal homology was found with a ferredoxin from Rhodo-pseudomonas palustris . Northern (RNA) hybridization using a 158-base-pair DNA fragment internal to the fdxN coding region revealed the existence of two mRNA transcripts of approximately 330 and 750 nucleotides . Neither of those transcripts was present under nif-repressing growth conditions . The 5' end of the smaller transcript was mapped by S1 nuclease protection and primer extension experiments . On the basis of Southern hybridization experiments, by using probes homologous to fdxN, nifE, and a fragment complementing a nif point mutation, fdxN was localized inside a cluster of nif genes.

Biochem Int, 1989 Nov, 19(5), 1125 - 31
Esterification of chiral secondary alcohols with fatty acid in organic solvents by polyethylene glycol-modified lipase; Kikkawa S et al.; Lipase from Pseudomonas fragi 22.39B was modified with polyethylene glycol . The modified lipase (PEG-lipase) was soluble and active in organic solvents such as benzene and 1,1,1-trichloroethane . PEG-lipase catalyzed esterification of chiral secondary alcohols with fatty acids in benzene and exhibited preference for R isomers over S isomers . Km and Vmax values for each isomer of various alcohols were obtained by kinetic study of the esterification in benzene . PEG-lipase-catalyzed esterification leads to optical resolution of a racemic alcohol.

Plasmid, 1989 Nov, 22(3), 271 - 4
Identification and DNA sequencing of a new plasmid (pPST1) in Pseudomonas stutzeri MO-19; Fujita M et al.; A cryptic plasmid, pPST1, was isolated from Pseudomonas stutzeri MO-19 and its complete nucleotide sequence was determined . This plasmid consisted of 1446 bp and could encode a putative polypeptide of 152 amino acid residues (ORF1) in an open reading frame . The putative protein contained a sequence homologous to the sequences found in DNA-binding sites.

Appl Environ Microbiol, 1989 Nov, 55(11), 3022 - 5
Detection in soil of a deletion in an engineered DNA sequence by using DNA probes; Jansson JK et al.; Two Pseudomonas strains were engineered to contain the nptII gene and plasmid vector sequences in their chromosomes . After incubation of these strains in nonsterile soil, total bacterial DNA was isolated and analyzed by Southern blot hybridization with the nptII gene and the plasmid vector as probes . In addition to the expected bands of hybridization, a new band corresponding to the loss of vector sequences from the chromosome while retaining the nptII gene was observed for one of the strains . The more stressful conditions encountered in soil appeared to increase the frequency of loss of the vector sequences from this strain.

Izv Akad Nauk SSSR Biol, 1989 Nov-Dec, (6), 916 - 21
{The plasmids of Pseudomonas syringae}; Boronin AM et al.; The paper deals with occurrence of plasmids in P . syringae strains belonging to seventeen pathovars: the strains were isolated in the USSR and other countries . One to four different plasmids having molecular weights of 20 to 90 Md have been found in various strains of the following twelve pathovars: holci, cerasi, aptata, tabaci, populi, pisi, lupini syringae, lachrymans, phaseolicola, glycinea, atrofaciens . Both virulent and avirulent P . syringae strains appear to carry such plasmids . Plasmids of incompatibility group P-2 do not occur in the strains studied.

Indian J Exp Biol, 1989 Nov, 27(11), 967 - 71
Metabolism of 3-chlorobenzoate by a Pseudomonas (diff) spp; Vora KA et al.; Pseudomonas (diff) spp . was isolated from a complex petrochemical sludge, using benzoate as the sole source of carbon . The organism could metabolize 3-chlorobenzoate, releasing approximately 30% of organically bound chloride . 3-Chlorodihydrodihydroxybenzoate and 3-chlorocatechol were confirmed as pathway intermediates by mass spectral and HPLC analysis . About 3-fold higher levels of catechol 1,2-oxygenase were detected in cells grown on 3-chlorobenzoate as compared to that of benzoate . 3-Chlorocatechol inhibited the catechol 1,2-oxygenase activity, when used as assay substrate . A 15-fold purified catechol 1,2-oxygenase had a Km of 0.37 mumole and Vmax of 2.3 with 3-chlorocatechol . Catechol gave Km of 0.2 mumole and Vmax of 40, suggesting that 3-chlorocatechol is not metabolised further and hence blocks the metabolic pathway for 3-chlorobenzoate degradation . In contrast catechol 1,2-oxygenase was not inhibited by 4-chlorocatechol and probably is an intermediate for the total/complete degradation of 3-chlorobenzoate (approx . 30%).

Rev Infect Dis, 1989 Nov-Dec, 11(6), 846 - 52
Pseudomonas bacteremia in a community teaching hospital, 1980-1984; Gallagher PG et al.; The epidemiologic characteristics, clinical course, and outcome of 96 episodes of pseudomonas bacteremia occurring during 1980-1984 in a community teaching hospital are reviewed . The male-to-female ratio for the patients involved was 1.9:1 . The mean age was 64 years, and 41 patients were greater than or equal to 70 years of age . Sixty-two episodes were nosocomially acquired; nine were acquired in nursing homes . Twenty episodes were polymicrobial . The respiratory and genitourinary tracts were the most common sources of bacteremia . Seven patients had splenectomies prior to the onset of bacteremia . The overall mortality was 61%; mortality attributable to pseudomonas bacteremia was 48% . Statistically significant differences in mortality were related to underlying conditions and portal of entry . Findings in this series were compared with those in published series from different institutions over a period of years . Pseudomonas bacteremia remains a major infection in patients with underlying diseases and continues to cause high mortality in the 1980s.

J Antimicrob Chemother, 1989 Nov, 24(5), 787 - 95
Clinical and pharmacokinetic aspects of ciprofloxacin in the treatment of acute exacerbations of pseudomonas infection in cystic fibrosis patients; Steen HJ et al.; Twelve cystic fibrosis patients, aged over 18, who had developed an acute respiratory exacerbation and who had Pseudomonas species isolated from their sputum, were entered into a clinical trial involving ciprofloxacin . The dosage regimen was 100 mg iv followed by 500 mg twice daily orally if less than 40 kg in weight and 200 mg iv followed by 750 mg twice daily orally if greater than 40 kg . Ciprofloxacin was well tolerated with no major side effects, except in one patient who withdrew after onset of headaches and generalized aches and pains . Eleven of the 12 patients showed clinical improvement at the end of the treatment period as determined by weight gain, Shwachman Score, Chrispin Norman Score and pulmonary function tests . MICs of Pseudomonas species isolated from the sputum at the start of the trial were in the range 0.25-4 mg/l . During therapy, sensitivity of isolates decreased and did not return to starting levels at the end of a four week follow-up period . Pharmacokinetic parameters were similar to those reported for fasting healthy volunteers by other workers except for bioavailability which was reduced in the non-fasting patients.

J Gen Microbiol, 1989 Nov, 135 ( Pt 11), 2909 - 15
Characterization of Pseudomonas mercury-resistance transposon Tn502, which has a preferred insertion site in RP1; Stanisich VA et al.; Tn502mer differs in size and restriction map from the well-characterized Tn501mer . It also differs in its preferential and high-frequency insertion into the 6 kb PstI-C region of RP1 . The affinity for this region is perpetuated in pVS76, a clone of RP1 PstI-C in pBR322 . Restriction mapping of independent pVS76::Tn502 derivatives revealed that Tn502 inserted at the same site (or small region) in PstI-C corresponding to the 35 kb coordinate in RP1 . Insertion occurred in both orientations, but one was preferred . When PstI-C was deleted from RP1, acquisition of Tn502 was reduced and the sites of insertion randomized.

J Inorg Biochem, 1989 Nov, 37(3), 233 - 57
Preparation and spectral characterization of the heme d1.apomyoglobin complex: an unusual protein environment for the substrate-binding heme of Pseudomonas cytochrome oxidase; Steup MB et al.; The heme d1 prosthetic group isolated from Pseudomonas cytochrome oxidase combines with apomyoglobin to form a stable, optically well-defined complex . Addition of ferric heme d1 quenches apomyoglobin tryptophan fluorescence suggesting association in a 1:1 molar ratio . Optical absorption maxima for heme d1.apomyoglobin are at 629 and 429 nm before, and 632 and 458 nm after dithionite reduction; they are distinct from those of heme d1 in aqueous solution but more similar to those unobscured by heme c in Pseudomonas cytochrome oxidase . Cyanide, carbon monoxide and imidazole alter the spectrum of heme d1.apomyoglobin demonstrating axial coordination to heme d1 by exogeneous ligands . The cyanide-induced optical difference spectra exhibit isosbestic points, and a Scatchard-like analysis yields a linear plot with an apparent dissociation constant of 4.2 X 10(-5) M . However, carbon monoxide induces two absorption spectra with Soret maxima at 454 or 467 nm, and this duplicity, along with a shoulder that correlates with the latter before binding, suggests multiple carbon monoxide and possibly heme d1 orientations within the globin . The 50-fold reduction in cyanide affinity over myoglobin is more consistent with altered heme pocket interactions than the intrinsic electronic differences between the two hemes . However, stability of the heme d1.apomyoglobin complex is verified further by the inability to separate heme d1 from globin during dialysis and column chromatography in excess cyanide or imidazole . This stability, together with a comparison between spectra of ligand-free and -bound derivatives of heme d1-apomyoglobin and heme d1 in solution, implies that the prosthetic group is coordinated in the heme pocket through a protein-donated, strong-field ligand . Furthermore, the visible spectrum of heme d1.apomyoglobin varies minimally with ligand exchange, in contrast to the Soret, which suggests that much spectral information concerning heme d1 coordination in the oxidase is lost by interference from heme c absorption bands . A comparison of the absorption spectra of heme d1.apomyoglobin and Pseudomonas cytochrome oxidase, together with a critical examination of the previous axial ligand assignments from magnetic resonance techniques in the latter, implies that it is premature to accept the assignment of bishistidine heme d1 coordination in oxidized, ligand-free oxidase and other iron-isobacteriochlorin-containing enzymes.

Infect Immun, 1989 Nov, 57(11), 3345 - 8
Pseudomonas elastase acts as a virulence factor in burned hosts by Hageman factor-dependent activation of the host kinin cascade; Holder IA et al.; Purified Pseudomonas elastase injected subcutaneously into the skin of an Evans blue dye-injected (intravenously) guinea pig caused dye leakage similar to that observed when histamine or bradykinin was injected in the same animal . The histamine-induced dye leakage was ablated in antihistamine-treated guinea pigs, but elastase- and bradykinin-induced dye leakages were not . Local injections of specific inhibitors of the host Hageman factor-dependent bradykinin-generating pathway given immediately prior to elastase injection reduced dye leakage in a dose-related manner . Elastase-related dye release was enhanced when angiotension-converting enzyme inhibitor, a substance which prevents host enzymes from breaking down bradykinin, was injected prior to elastase injection . We conclude that Pseudomonas elastase generates bradykinin in the infected host via a Hageman factor-dependent pathway.

Cancer Res, 1989 Nov 1, 49(21), 6019 - 23
Enhancement of trimetrexate cytotoxicity in vitro and in vivo by carboxypeptidase G2; Romanini A et al.; Carboxypeptidase G2 (CPG2), an enzyme produced by Pseudomonas strain RS-16, hydrolyzes the glutamate residue from methotrexate and other folates . The possibility of enhancing trimetrexate cytotoxicity by CPG2 induced folate depletion was investigated in vitro in a human leukemia cell line, CCRF-CEM, and in three sublines of these cells each with a different methotrexate resistance phenotype . The cytotoxic effect in vitro was detected using a colorimetric assay with a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide . Dose-effect relationships of drugs alone and in combination were analyzed by the median effect principle and by the combination indices for quantitation of synergy or antagonism with the aid of a computer program . Trimetrexate alone was cytotoxic against the parent and all the resistant cell lines with the drug concentrations required to decrease the cell count to 50% of control in the nanomolar range (1.4, 1.6, 1.5, and 0.7 nM in CCRF-CEM, CCRF-CEM/E, CCRF-CEM/P, and CCRF-CEM/T, respectively) following 5 days of exposure . The concentration of CPG2 required to decrease the cell count to 50% control for these cell lines was 3.5, 2.6, 26.6, and 7.9 x 10(-5) units/ml for CCRF-CEM, CCRF-CEM/E, CCRF-CEM/P, and CCRF-CEM/T, respectively . A synergistic cytotoxic effect of trimetrexate after simultaneous continuous exposure with CPG2 was observed with CCRF-CEM cells and with the three resistant cell lines . This drug combination given to BALB/c x DBA/2 F1 mice bearing L1210 cells also produced synergy over a narrow range of drug doses . The activity of this combination in both methotrexate sensitive and methotrexate resistant cell lines indicates that clinical trials of this combination should be undertaken.

Proc Natl Acad Sci U S A, 1989 Nov, 86(21), 8545 - 9
Antitumor activity in mice of an immunotoxin made with anti-transferrin receptor and a recombinant form of Pseudomonas exotoxin; Batra JK et al.; LysPE40 is a modified form of Pseudomonas exotoxin that lacks the cell-binding domain and has a chemically reactive lysine residue near the amino terminus . LysPE40 is made in Escherichia coli and secreted into the medium from which it is readily purified . Two immunotoxins were constructed by coupling LysPE40 to an antibody to the human transferrin receptor (TFR) or to an antibody to the human interleukin-2 receptor . These immunotoxins were selectively cytotoxic to receptor-bearing cells in tissue culture . Anti-TFR-LysPE40 given intraperitoneally to mice appeared rapidly in the blood and caused regression of A431 tumors growing as subcutaneous xenografts . These results show that it is possible to cause regression of a solid carcinoma by an immunotoxin if proper targeting can be achieved.

J Biol Chem, 1989 Oct 25, 264(30), 17919 - 23
Role of cysteine residues in Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA reductase . Site-directed mutagenesis and characterization of the mutant enzymes; Jordan-Starck TC et al.; Each of the four identical subunits of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase contains two cysteine residues, Cys156 and Cys296 (Beach, M . J., and Rodwell, V . W . (1989) J . Bacteriol . 171, 2994-3001) . Both are accessible to modification by sulfhydryl reagents under nondenaturing conditions (Jordan-Starck, T . C., and Rodwell, V . W . (1989) J . Biol . Chem . 264, 17913-17918) . We used site-directed mutagenesis to construct three mutant enzymes in which alanine replaced either or both cysteine residues . Mutant enzymes C156A, C296A, and C156/296A were over-expressed in Escherichia coli and were found to be fully active . Following their purification, all four forms of the enzyme were compared with respect to their catalytic efficiency, their affinities for the substrates of all four catalyzed reactions, and for their sensitivity to inactivation by sulfhydryl reagents . Replacement of cysteine residues with alanine residues had no major effect on either the specific activity or the affinity of the enzymes for any substrate . The mutants catalyzed all four HMG-CoA reductase reactions as efficiently as did the wild-type enzyme, and coenzyme A stimulated mevaldehyde reduction to the same extent as for wild-type HMG-CoA reductase . Mutant C156A and the cysteine-free mutant C156/296A were not inactivated by 5,5'-dithiobis(2-nitrobenzoate) . By contrast, mutant C296A was inactivated to the same extent as was the wild-type enzyme . Following treatment of the mutant enzymes with N-ethylmaleimide, the four reductase reactions catalyzed by mutant C296A were inactivated to the same extent as for the wild-type enzyme . Neither mutant C156A nor C156/296A was affected by this reagent . We conclude that the sulfhydryl reagent-reactive group whose derivatization leads to loss of enzymatic activity is Cys156 . However, this residue is not an essential active site residue since neither substrate binding nor catalysis was affected when it was replaced by alanine . Possible roles of cysteine in maintaining structural stability are discussed.

J Biol Chem, 1989 Oct 25, 264(30), 17913 - 8
Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA reductase . Characterization and chemical modification; Jordan-Starck TC et al.; Pseudomonas mevalonii (formerly designated Pseudomonas sp . M (Beach, M . J., and Rodwell, V . W . (1989) J . Bacteriol . 171, 2994-3001; Gill, J . F., Jr., Beach, M.J., and Rodwell, V . W . (1985) J . Biol . Chem . 260, 9393-9398} 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.88), overexpressed in Escherichia coli (1), has been purified to electrophoretic homogeneity in 75% yield (final specific activity 48 mumols of NAD+ reduced per min/mg protein) . The enzyme catalyzes its normal catabolic reaction (mevalonate + 2 NAD+ + CoASH----HMG-CoA + 2NADH + 2H+), and two half-reactions which involve mevaldehyde, the postulated intermediate in the aforementioned reactions and mevaldehyde + NADH + H+----mevalonate + NAD+) . The rates of all four reactions and the Michaelis constants for all substrates were measured . Coenzyme A decreased the KM for mevaldehyde reduction 12-fold and stimulated VMAX 2-3 fold . CoASH thus may remain bound throughout the catalytic cycle . Dithiothreitol and analogs of CoASH were tested for their ability to reproduce the CoASH stimulation . Pantetheine, but not dithiothreitol, pantothenate, or desulfo-CoA mimicked CoASH stimulation . Titration with 5,5'-dithiobis(2-nitrobenzoic acid) indicated two sulfhydryl groups per subunit . Both groups remained accessible to 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of mevalonate and/or NAD+ but only one group in the presence of HMG-CoA . N-Ethylmaleimide inhibited all the aforementioned reactions . HMG-CoA, but not mevalonate, afforded protection completely and irreversibly inactivated the enzyme . The reactive sulfhydryl group thus may not be a catalytic residue, but may be involved in a conformational change.

J Natl Cancer Inst, 1989 Oct 4, 81(19), 1455 - 63
Targeted toxin therapy for the treatment of cancer; FitzGerald D et al.; Protein toxins such as Pseudomonas exotoxin, diphtheria toxin, and ricin may be useful in cancer therapy because they are among the most potent cell-killing agents . One molecule of a toxin delivered to the cytoplasm of a cancer cell will be lethal for that cell . However, to be therapeutically useful, these toxins need to be targeted to specific sites on the surface of cancer cells, then be internalized and ultimately reach the cell cytoplasm . This process is accomplished by eliminating binding to toxin receptors and redirecting the cell-killing activity of the toxin to receptors or antigens present on cancer cells . Typically, toxins are conjugated to cell-binding proteins such as monoclonal antibodies or growth factors . These conjugates bind and kill cancer cells selectively while normal cells, which don't bind the conjugates, are spared . Because the genes for many protein toxins have been cloned, it is possible to make genetic modifications to their structure . By deleting the DNA that codes for the toxin binding region and replacing it with various complementary DNA encoding other cell-binding proteins, it has been possible to make chimeric toxins that kill cells on the basis of the newly acquired binding activity . The ability to make these chimeras may be useful in designing future toxin-based anticancer therapies.

CLAO J, 1989 Oct-Dec, 15(4), 264 - 5
Pseudomonas corneal ulcer associated with disposable soft contact lenses; Kent HD et al.; Disposable contact lenses were developed to reduce the complications associated with cosmetic extended wear contact lenses . We describe two cases of Pseudomonas corneal ulcers that developed in patients wearing disposable soft contact lenses . Patients wearing these lenses remain at risk for infection.

Zentralbl Veterinarmed B, 1989 Oct, 36(8), 639 - 40
Growth inhibition of Escherichia coli by nitrate reductase; Tamasi G et al.; The metabolite of a not nearer identified Pseudomonas strain inhibited or inactivated Escherichia coli in presence of nitrate.

Br J Clin Pract, 1989 Oct, 43(10), 383 - 5
Osteomyelitis of the symphysis pubis: a complication of cardiac catheterisation; Guthrie R et al.; An elderly female experienced the onset of right femoral pain shortly after undergoing cardiac catheterisation . Her condition was later found to be Pseudomonas osteomyelitis of the symphysis pubis . The patient did not have any of the usual risk factors for Pseudomonas osteomyelitis, ie, intravenous drug abuse, contiguous surgery or trauma . The probable mechanism of bacteria seeding into a haematoma around the femoral artery and then spreading to the contiguous osseus structures is proposed for this unusual complication.

Res Microbiol, 1989 Oct, 140(8), 553 - 62
Role of transmembrane electrical potential on cadmium fixation by a marine pseudomonad; Flatau GN et al.; The role of cellular energy, and mainly that of electrical transmembrane potential, in cadmium fixation by a marine pseudomonad suspended in a mineral medium was investigated by studying the effects of ionophores . Although fixation of cadmium by cells was generally less when respiratory activity was inhibited, it was not affected by a reduction of the transmembrane electrical potential delta psi in mureinoplasts . These observations strongly suggest that cadmium fixation in this isolate was not the result of a delta psi-dependent active transport.

Kansenshogaku Zasshi, 1989 Oct, 63(10), 1160 - 4
{A strain of Pseudomonas vesicularis isolated from shower hose which supports the multiplication of Legionella}; Koide M et al.; In Japan, a fatal case due to Legionella micdadei was first recognized in our laboratory in 1986 . On the epidemiological study just after the case, no Legionella was detected from the environmental samples of the patient's residence, such as shower water, tank water and so on . In the course of prospective investigations, no Legionella was isolated, but many organisms were grown on BCYE alpha and MWY agar plates . In the retrospective study, one of these organisms was found to support satellite growth of Legionella on BCYEagar without L-cysteine . This was the isolate from the shower hose and identified as Pseudomonas vesicularis with the biochemical and DNA-DNA hybridization test . And P . vesicularis type strain ATCC11426 also supported satellite growth of Legionella . Especially in the water supply system, the existence of P . vesicularis seemed to be effective on the growth of Legionella . It must be taken into consideration that efforts made to isolate the nutrient produced organisms as well as Legionella are needed.

J Appl Bacteriol, 1989 Oct, 67(4), 377 - 93
Identification of psychrotrophic pseudomonads from goats' milk by computer-assisted analysis of carbon source assimilation data; Cox JM et al.; The results of carbon source assimilation tests on a group of psychrotrophic pseudomonas were compared with published data for established Pseudomonas taxa, using computer-assisted numerical taxonomic analysis and a modified diagnostic computer program . Several phenons were not grouped at the biovar level by numerical taxonomic analysis . Identification of strains by the diagnostic program revealed heterogeneity among those in the unassigned phenons, and supported a continuum concept among the fluorescent pseudomonads.

Biokhimiia, 1989 Oct, 54(10), 1658 - 65
{Study of the inhibition of neutral phosphatase from Pseudomonadaceae by derivatives of its substrates}; Krupianko VI et al.; The inhibition of neutral phosphatase isolated from the bacteria of the Pseudomonadaceae family by various fragments of the enzyme-hydrolyzed R-O-PO3H2 substrates, inorganic orthophosphate (KH2PO4) and its analogs as well as by adenine, adenosine, alcohols, sugars and amino acids, was studied . It was demonstrated that among other compounds tested only the orthophosphoric acid anions (H2PO4-) exhibit the properties of strong associative inhibitors (K1Vi = 4.35.10(-6)M of the enzyme . The pH dependence of the Michaelis constant {pKm0 = f(pH)} and the inhibition constant for phosphatase by potassium orthophosphate {pK1Vi(KH2PO4) = f(pH)} was studied . The presence in the enzyme active center of a carboxylic (pK = 4.3 +/- 0.1) (presumably, glutamine) and an imidazole (pK = 7.15 +/- 0.1) amino acid residues was postulated . The data obtained were compared to those for neutral, alkaline and acid phosphatases.

J Bacteriol, 1989 Oct, 171(10), 5567 - 71
(S)-3-hydroxy-3-methylglutaryl coenzyme A reductase, a product of the mva operon of Pseudomonas mevalonii, is regulated at the transcriptional level; Wang YL et al.; We have cloned and sequenced a 505-base-pair (bp) segment of DNA situated upstream of mvaA, the structural gene for (S)-3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.88) of Pseudomonas mevalonii . The DNA segment that we characterized includes the promoter region for the mva operon . Nuclease S1 mapping and primer extension analysis showed that mvaA is the promoter-proximal gene of the mva operon . Transcription initiates at -56 bp relative to the first A (+1) of the translation start site . Transcription in vivo was induced by mevalonate . Structural features of the mva promoter region include an 80-bp A + T-rich region, and -12, -24 consensus sequences that resemble sequences of sigma 54 promoters in enteric organisms . The relative amplitudes of catalytic activity, enzyme protein, and mvaA mRNA are consistent with a model of regulation of this operon at the transcriptional level.

J Biol Chem, 1989 Sep 25, 264(27), 15953 - 9
Domain II mutants of Pseudomonas exotoxin deficient in translocation; Jinno Y et al.; Pseudomonas exotoxin (PE) kills mammalian cells in a complex process that involves cell surface binding, internalization by endocytosis, translocation to the cytosol, and ADP-ribosylation of elongation factor 2 . PE is a three-domain protein in which domain I binds to the cell surface, domain II promotes translocation into the cytosol, and domain III carries out ADP-ribosylation . To determine how translocation occurs, we have mutated all the arginine residues in domain II and found that mutations at positions 276 and 279 greatly diminished the cytotoxicity of PE and mutations 330 and 337 substantially reduced cytotoxicity . Biochemical studies indicate that after internalization into an endocytic compartment, the PE molecule undergoes a specific and saturable intracellular interaction, and this interaction is deficient in an Arg276----Gly mutant . Our data suggest that the translocation process of PE involves a specific interaction of Arg276 (and possibly Arg279, Arg330, and Arg337) with components of an intracellular compartment.

What Is MIC?, What Is Environmental Microbiology?, What Is Functional Genomics?, What Is Protein?, What Is Water Purification?, r, Bacteria, r, Microbes, a, Microbe, i, Microorganism, s, Bacteriology, e, S. cerevisiae, a, Fermentations, e, Fermentations, n, Escherichia coli, r, Fermentations, o, Cell cultures, i, Vibriosis, o, Bactericidal, s, Propionibacter, i, Microbiological, a, Staphylococcus, n, Microorganism, c, Erythromycin, a, Escherichia coli, o, Escherichia coli, i, Proteus, s, Paenibacilli, r, Staphylococcus aureus, n, Escherichia coli, s, Salmonella, r, Escherichia coli

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005