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