Pseudomonas

Protein Eng, 1994 Apr, 7(4), 559 - 62
Analysis of the structure of Pseudomonas glumae lipase; Noble ME et al.; The lipase produced by Pseudomonas glumae is monomeric in the crystalline state and has a serine protease-like catalytic triad; Ser87-His285-Asp263 . The largest domain of the protein resembles closely a subset of the frequently observed alpha/beta-hydrolase fold and contains a well-defined calcium site . This paper describes structural analysis of this protein, focusing on (i) structural comparison with the lipase from Geotrichum candidum, (ii) the probable nature of the conformational change involved in substrate binding and (iii) structural variations amongst the family of Pseudomonas lipases . This analysis reveals similarities between P . glumae lipase and G . candidum lipase involving secondary structural elements of the hydrolase core and the loops carrying the catalytic serine and histidine residues . A possible functional equivalence has also been identified between parts of the two molecules thought to be involved in a conformational change . In addition, determination of the structure of P . glumae lipase has allowed rationalization of previously reported protein engineering experiments, which succeeded in improving the stability of the enzyme with respect to proteolysis.

Appl Environ Microbiol, 1994 Apr, 60(4), 1121 - 8
Analysis of competition in soil among 2,4-dichlorophenoxyacetic acid-degrading bacteria; Ka JO et al.; Competition among indigenous and inoculated 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was studied in a native Kansas prairie soil following 2,4-D additions . The soil was inoculated with four different 2,4-D-degrading strains at densities of 10(3) cells per g of soil; the organisms used were Pseudomonas cepacia DBO1(pJP4) and three Michigan soil isolates, strain 745, Sphingomonas paucimobilis 1443, and Pseudomonas pickettii 712 . Following 2,4-D additions, total soil DNA was extracted and analyzed on Southern blots by using a tfdA gene probe which detected three of the strains and another probe that detected the fourth strain, S . paucimobilis 1443, which belongs to a different class of 2,4-D degraders . P . cepacia DBO1(pJP4), a constructed strain, outcompeted the other added strains and the indigenous 2,4-D-degrading populations . The S . paucimobilis population was the secondary dominant population, and strain 745 and P . pickettii were not detected . Relative fitness coefficients determined in axenic broth cultures predicted the outcome of competition in soil for some but not all strains . Lag time was shown to be a principal determinant of competitiveness among the strains, but the lag times were significantly reduced in mixed broth cultures, which changed the competitive outcome . Plasmids containing the genes for the 2,4-D pathway were important determinants of competitiveness since plasmid pKA4 in P . cepacia DBO1 resulted in the slower growth characteristic of its original host, P . pickettii, rather than the rapid growth observed when this strain harbors pJP4.

Neurosurgery, 1994 Apr, 34(4), 649 - 55; discussion 655-6
In vivo efficacy of intrathecal transferrin-Pseudomonas exotoxin A immunotoxin against LOX melanoma; Hall WA et al.; Neoplastic meningitis due to the dissemination of systemic cancer or primary central nervous system tumors through the cerebrospinal fluid carries a very poor prognosis . Current treatments for this disease are ineffective, and new therapeutic modalities such as immunotoxins may be beneficial . We created an animal model of human carcinomatous meningitis with LOX melanoma-derived tissue-culture cells in athymic rats for testing the efficacy of intrathecal therapy with transferrin-Pseudomonas exotoxin A (Tfn-PE) immunotoxin . An injection of 5 x 10(5) LOX cells into the intrathecal space through an indwelling catheter resulted in the reproducible development of lower-extremity paraplegia at 9.24 +/- 1.77 days because of focal deposits of tumor growth adjacent to the thoracic and lumbar spinal cord . A dose of 2.5 or 5 micrograms of intrathecal Tfn-PE immunotoxin was neurotoxic and resulted in the deaths of 8 of 10 animals within 24 hours . Histological evidence of central nervous system damage was seen as hemorrhagic degeneration around the central canal or a pathological cleft at the level of the cervical spinal cord . Because no neurotoxicity was seen with 1 microgram of intrathecal Tfn-PE immunotoxin, this dose was administered in treatment experiments . Twenty-four hours after the intrathecal instillation of LOX cells, 10 animals received intrathecally either 1 microgram of Tfn-PE or phosphate-buffered saline with 0.1% human serum albumin (control group).(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1994 Apr, 60(4), 1093 - 1100
Restriction fragment length polymorphism evidence for genetic homology within a pathovar of Pseudomonas syringae; Scholz BK et al.; Pseudomonas syringae pv . phaseolicola NPS3121 hrp sequences were used as hybridization probes in a restriction fragment length polymorphism (RFLP) analysis of 24 P . syringae pv . tabaci strains as a means to evaluate the genetic and taxonomic relationship of pathovars of P . syringae . Southern blot analyses of genomic restriction digests, with hrpA-S sequences as hybridization probes, and restriction analyses of PCR-amplified DNA of regions within hrpD were conducted . The resulting RFLP patterns were uniform for 23 of the 24 isolates tested, with strain BR2R having a unique pattern . BR2R is a pathogen of bean which was classified as pathovar tabaci because of its ability to produce tabtoxin, but unlike the other 23 tabaci strains in this study, it does not incite disease symptoms on tobacco . When a DNA fragment containing hrpM sequences was used as a hybridization probe, the tabaci isolates could be divided into three groups on the basis of the RFLP patterns : BR2R, Pt11528R and Pt113R, and the remaining strains . For all of the above analyses, BR2R shared identical RFLP patterns with P . syringae pv . phaseolicola NPS3121, also a bean pathogen which does not cause disease on tobacco . However, BR2R AND NPS3121 could be differentiated from each other on the basis of the RFLP patterns from restriction analysis of PCR-amplified DNA of argF, while the remaining tabaci strains had a third pattern . These studies indicate that hrp genes and argF are conserved in strains of P . syringae pathogenic to tobacco, suggesting that P . syringae strains pathogenic to specific hosts may have a high level of genetic similarity . We believe that these analyses have shown that distinct identifiable genetic differences may be correlated with host range and suggest that such information may be useful for assigning pathovar designations.

Biosci Biotechnol Biochem, 1994 Apr, 58(4), 752 - 5
Purification and characterization of a carboxylesterase from Pseudomonas sp . KWI-56; Sugihara A et al.; An intracellular carboxylesterase from Pseudomonas sp . was overproduced in E . coli, and purified to homogeneity by a combination of hydrogen bond chromatography, gel filtration, and hydrophobic interaction chromatography . Gel filtration and SDS-PAGE suggested that the purified enzyme consisted of two subunits of molecular mass of 28 kDa . Its isoelectric point was 5.9 . The enzyme was thermolabile, and showed its maximum activity at 22 degrees C (pH 7.5) . Methyl propionate was hydrolyzed at the highest rate among the fatty acid methyl esters tested . PMSF, DFP, PCMB, and HgCl2 inhibited the enzyme markedly, suggesting that serine and/or cysteine is in or near the active site.

J Clin Microbiol, 1994 Apr, 32(4), 924 - 30
Linkage analysis of geographic and clinical clusters in Pseudomonas cepacia infections by multilocus enzyme electrophoresis and ribotyping; Johnson WM et al.; Multilocus enzyme electrophoresis and ribotyping were used to characterize 83 strains of Pseudomonas cepacia, mostly isolated from cystic fibrosis (CF) patients, although a number of isolates from non-CF nosocomial infections and reference environmental strains were represented . Twenty enzyme electrophoretic types (ETs) were determined; of these, one clone (ET12) was associated with six of nine ribotypes (RTs) said to be geographically representative of the United Kingdom and all of the Ontario (Canada) isolates from CF patients . This clone was not associated with nosocomial infections or environmental strains and was never found in CF isolates from British Columbia or Nova Scotia, Canada, or a center in the eastern United States . Individual isolate EcoRI RT signatures did not cluster geographically as did the ET signatures by clonal analysis . Frequently RTs occurred in more than a single ET . Known point source focal nosocomial outbreaks were typified by single ETs and stable RTs . Dendrographic analysis of the strains grouped those strains from CF patients, nosocomial outbreaks, and environmental sources into separate ET families, and diversity analysis indicated that, with the exception of ET17, CF isolates clustered in unique and closely related ETs different from those from nosocomial and environmental sources . This study has also shown the potential of multilocus enzyme electrophoresis to monitor the intercontinental spread of P . cepacia strains in CF patients, and this may have a significant impact on plans for CF patient summer camps and design of infection control practices . Whether the intercontinental ET12 clone, which predominates in the United Kingdom and the province of Ontario, linked by summer camp acquisition, has increased virulence for CF patients remains to be established.

Transplantation, 1994 Mar 27, 57(6), 823 - 6
Retransplantation in hepatitis B--a multicenter experience; Crippin J et al.; Hepatitis B has become one of the most challenging diseases in liver transplantation . Infection of the allograft with subsequent graft failure is common and may prompt consideration of repeat liver transplantation . The aims of this study were to examine the experience in the United States with retransplantation in hepatitis B patients with recurrent disease as well as for other reasons . Questionnaires were mailed to adult liver transplant centers in the United States, requesting information on retransplantation in HBV patients . Responses were received from 71% of the centers . Thirty-eight patients were retransplanted, 20 for recurrent HBV and 18 for other reasons . The survival rate following retransplantation for HBV was poor . Nine patients (55%) died within 60 days . Eleven patients survived 60 days or longer, though eight died at a mean of 4.1 +/- 2 months, one required a third transplant for recurrent HBV infection at 4 months, and one died at 35 months . Only a single (5%) long-term survivor exists . Recurrent histologic disease occurred earlier in the second transplant at 2.8 +/- 2.9 months versus 6.1 +/- 5.2 months in the first allograft, though this difference did not reach statistical significance (P = .058) . Patients transplanted for other reasons (primary non-function {9}, hepatic artery thrombosis {6}, persistent rejection {2}, and a Pseudomonas graft infection {1}) had a better survival rate . Four patients survived less than 60 days . Of the 14 surviving longer than 60 days, 11 patients are alive at a mean of 21.2 +/- 14.8 months . Retransplantation for recurrent HBV appears to be contraindicated due to a high mortality . Retransplantation in HBV patients with graft failure due to other causes, however, should be considered, since over 60% of these patients appear to have good long-term survival . Additional studies examining risk factors for recurrent disease should be considered.

J Biol Chem, 1994 Mar 11, 269(10), 7610 - 6
Pseudomonas exotoxin A mutants . Replacement of surface exposed residues in domain II with cysteine residues that can be modified with polyethylene glycol in a site-specific manner; Kuan CT et al.; Pseudomonas exotoxin A (PE) is a three-domain protein in which domain Ia is involved in recognition of receptors on eukaryotic target cells, domain II promotes translocation of PE into the cytosol, and domain III enzymatically ADP-ribosylates elongation factor 2 . Modification of proteins with polyethylene glycol (PEG) has been shown to prolong circulating plasma lifetime and may reduce or eliminate immunogenicity . However, in the case of toxins, PEG may interfere with or block toxin activity . To investigate the effect of polyethylene glycolation on specific residues located on the surface of PE domain II, we substituted cysteine, for each of the five most exposed surface amino acids (H276, E282, N306, R313, and E327) in domain II . These cysteines can serve as unique sites for PEG modification . The PE-Cys proteins retained most of their cytotoxicity even when the free sulfhydryl group was blocked by 5,5'-dithiobis(nitrobenzoic acid) or glutathione . When the PE-Cys proteins were conjugated with ovalbumin using a cleavable disulfide linkage, cytotoxicity was retained, but it was lost with a non-cleavable thioether linkage . In contrast, cytotoxicity was maintained when PE-Cys mutants were coupled to 5- or 20-kDa mPEG, using either a disulfide or a thioether linkage . Unexpectedly in some cases, the thioether conjugate was more active than the disulfide linkage . Pharmacokinetic studies on one of the polyethylene-glycolated molecules (R313C) showed that the mean residence time (t 1/2) was prolonged to 72 min, compared to 20 min for unpolyethylene glycolated PE-Cys(R313C) . These studies show it is possible to derivatize PE at specific residues in domain II, maintain significant cytotoxic activity, and alter pharmacokinetics . These studies also suggest that large mPEG molecules can be translocated to the cytosol while still attached to domain II of PE.

Biochemistry, 1994 Mar 8, 33(9), 2682 - 7
Mechanism of the reaction catalyzed by delta 5-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: kinetic properties of a modified enzyme in which tyrosine 14 is replaced by 3-fluorotyrosine; Brooks B et al.; Tyrosine 14 of delta 5-3-ketosteroid isomerase plays an important role in the function of the enzyme, since its replacement by phenylalanine results in a decrease in kcat by a factor of 10(-4.7) . This result and the fact that this residue resides in the enzyme's substrate binding site and is in close proximity to C-2 of the bound steroid suggests that it functions as an electrophile in the catalytic mechanism by protonation of or hydrogen bonding to the C-3 carbonyl oxygen of the substrate . In order to obtain more information about the role of tyrosine 14, we have prepared a modified form of the enzyme in which tyrosine 14 has been substantially replaced in vivo by exogenously supplied 3-fluorotyrosine, a tyrosine derivative in which the pKa' of the phenol hydroxyl should be decreased by about 1.5 log units . Site specificity of this modification has been ensured by mutation of the codons for the nonessential tyrosines 55 and 88 to phenylalanine . We find that replacement of tyrosine 14 by 3-fluorotyrosine in the Y55,88F modified form of the isomerase results in a 4-fold decrease in kcat . We interpret this result in terms of a mechanism in which the transition state for enolization is dienolate-like, characterized by relatively little proton transfer from tyrosine 14 in the transition state, and the intermediate in the overall reaction is dienol-like . An alternative mechanism in which the intermediate is stabilized by a short, strong hydrogen bond can also be consistent with the data.

Biochemistry, 1994 Mar 8, 33(9), 2672 - 81
Extent of proton transfer in the transition states of the reaction catalyzed by the delta 5-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: site-specific replacement of the active site base, aspartate 38, by the weaker base alanine-3-sulfinate; Holman CM et al.; Previous studies of the mechanism of the steroid isomerase of Comamonas (Pseudomonas) testosteroni have identified aspartate 38 as the proton porter which transfers the substrate's 4 beta proton to the 6 beta position of the product . Consequently, aspartate 38 functions as a base in the deprotonation of the substrate to form a dienol or dienolate intermediate, which then undergoes reprotonation from protonated aspartate 38 at C-6 beta to give the product . We have tried to characterize the transition states for the proton transfers by altering the pKa' of aspartate 38 and then determining the effect of the alteration on the kinetics of the enzyme . Alteration of the pKa' was accomplished by replacement of the carboxyl carbon of aspartate 38 by sulfur, a change which converts the carboxylate group to the much less basic sulfinate group . Employing Bronsted catalysis theory as applied to the individual steps of the isomerase mechanism, we find that in the enolization step of the reaction proton transfer to aspartate 38 is well advanced in the transition state . In the subsequent ketonization step, proton transfer from aspartate 38 has barely started when that transition state is reached . A series of mutant KSIs with alternative bases at position 38 have been constructed using a combination of site-directed mutagenesis and chemical modification: Asp-38 to Glu (D38E), His (D38H), and S-(carboxymethyl)cysteine (D38CMC) . While the D38H and D38E mutants both retain significant isomerase activity, D38CMC is essentially inert . From the results of kinetic experiments it is possible to get a qualitative idea of the sensitivity of the enzyme's catalytic ability to the location of the base responsible for proton transfer.

Biochemistry, 1994 Mar 8, 33(9), 2620 - 7
Evidence for an equilibrium intermediate in the folding-unfolding pathway of a transforming growth factor-alpha-Pseudomonas exotoxin hybrid protein; Gress JO et al.; TP40 is a chimeric protein containing transforming growth factor-alpha (TGF-alpha) at the N-terminus and a Cys-->Ala mutant (PE40 delta Cys) of a 40,000-dalton segment (PE40) of Pseudomonas exotoxin (PE) . The guanidine hydrochloride (Gdn-HCl)-induced unfolding of TP40 and PE40 delta Cys has been studied by tryptophan fluorescence, circular dichroism (CD), and high-performance size exclusion chromatography (HPSEC) . The equilibrium unfolding of both proteins involves at least one intermediate (I) . In the I state(s), which may be induced by 1.3-2.0 M Gdn-HCl, the tertiary structure is fully or partially collapsed as detected by tryptophan fluorescence and near-UV CD, but the protein largely retains the native secondary structure and a semicompact shape as judged by far-UV CD and HPSEC, respectively . Soluble aggregates of TP40 and PE40 delta Cys are observed in addition to monomers at these intermediate (but not at higher) Gdn-HCl concentrations, suggesting that self-association is possibly mediated by thermodynamically stable, partially unfolded I states . The kinetics of refolding of TP40 upon dilution of Gdn-HCl involve two or more phases . Re-formation of secondary structure occurs rapidly (t 1/2 < 10 s) as determined by CD and is followed by a biphasic refolding of the native tertiary structure as detected by changes in tryptophan fluorescence . The midpoint (Tm) of the thermal unfolding transition occurs at a lower temperature when measured by tryptophan fluorescence than when detected by DSC and CD . These data suggest that Gdn-HCl and temperature can induce conformation(s) of TP40 that are distinct from native (N) and unfolded (U) states.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1994 Mar 4, 236(4), 1169 - 85
Crystal structure and refinement of cytochrome P450terp at 2.3 A resolution; Hasemann CA et al.; Cytochrome P450terp is a class I (mitochondrial/bacterial) P450 that catalyzes the hydroxylation of alpha-terpineol as part of the catabolic assimilation of this compound by a pseudomonad species . Crystals grown from the purified protein have the symmetry of space group P6(1)22, and cell dimensions a = b = 69.4 A, c = 456.6 A, alpha = beta = 90 degrees, gamma = 120 degrees . Diffraction data were collected at the Cornell High Energy Synchrotron Source, and the structure of P450terp was solved by a combination of molecular replacement and multiple isomorphous replacement techniques . A model of P450terp was built and refined against native data, to an R-factor of 18.9% for data with I > or = sigma(I) between 6.0 A and 2.3 A resolution . This model contains 412 of the 428 P450terp amino acid residues; the loop between helices F and G is disordered in the crystal . While the overall fold of P450terp is very similar to that of P450cam, only three-quarters of the C alpha positions can be superimposed, to a root-mean-square deviation of only 1.87 A . The mode of substrate binding by P450terp can be predicted, and probable substrate contact residues identified . The heme environment and side-chain positions in the adjacent I-helix suggest possible modes of proton delivery in the catalytic cycle of the enzyme.

Appl Environ Microbiol, 1994 Mar, 60(3), 966 - 72
Comparative biochemical and genetic analysis of naphthalene degradation among Pseudomonas stutzeri strains; Rossello-Mora RA et al.; Of a 49-strain collection of Pseudomonas stutzeri species, 11 isolates were able to degrade naphthalene and 1 isolate was able to use m- and p-toluate as sole carbon and energy sources . Of these 12 strains, 10 shared a highly homologous set of naphthalene catabolic genes, even though they belong to four different genomovars . These genes differed from those present in plasmid NAH7 . In only one of these degraders could a plasmid-encoded pathway be demonstrated, and a chromosome-encoded pathway is proposed for the remaining strains . meta cleavage of catechol was only observed in those strains able to metabolize alkyl derivatives of catechol.

J Bacteriol, 1994 Mar, 176(6), 1689 - 94
Identification of the bphA4 gene encoding ferredoxin reductase involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp . strain KKS102; Kikuchi Y et al.; The nucleotide sequence of the downstream region of the bph operon from Pseudomonas sp . strain KKS102 was determined . Two open reading frames (ORF1 and ORF2) were found in this region, and the deduced amino acid sequence of ORF2 showed homology with the sequences of four ferredoxin reductases of dioxygenase systems . When this region was inserted just upstream of the bph operon, which does not contain a gene encoding ferredoxin reductase, biphenyl dioxygenase activity was detected . The 24- and 44-kDa polypeptides predicted from the two open reading frames were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Crude extract which contained the products of ORF2 and bphA1A2A3 showed cytochrome c reduction activity . These data clearly suggest that ORF2 encodes ferredoxin reductase . The deduced amino acid sequence of ORF1 does not show significant homology with the sequences of any other proteins in the SWISS-PROT data bank, and the function of ORF1 is unknown.

J Bacteriol, 1994 Mar, 176(5), 1374 - 82
A mutation in the indole-3-acetic acid biosynthesis pathway of Pseudomonas syringae pv . syringae affects growth in Phaseolus vulgaris and syringomycin production; Mazzola M et al.; Homologs of the genes for indole-3-acetic acid (IAA) biosynthesis from Pseudomonas syringae pv . savastanoi were retrieved from a genomic library of P . syringae pv . syringae, and their nucleotide sequences were determined . Sequence relatedness between the P . syringae pv . syringae and P . syringae pv . savastanoi iaa operons is greater than 90% within the iaaM and iaaH loci but declines dramatically at a position approximately 200 bp 5' of the iaaM translation initiation codon . A third open reading frame was detected downstream of iaaH . Production of IAA was undetectable in mutant strain Y30-53.29, which was generated by transposition of Tn5 into the iaaM gene of P . syringae pv . syringae Y30 . The IAA-deficient (IAA-) mutant retained the ability to colonize the bean phylloplane and induced disease symptoms on bean which were similar to those produced by the parental strain . However, the population dynamics of the IAA- strain during the parasitic phase in leaves differed from those of both the parental strain and the mutant genetically restored for IAA biosynthesis . The mutant was capable of inducing disease symptoms when established in bean tissues at a lower initial cell density than either IAA-producing strain . Syringomycin biosynthesis by the IAA- strain was diminished in comparison with the parental strain or the mutant genetically restored for IAA production . The results indicate that bacterially derived IAA, or its biosynthesis, is involved in the regulation of in planta growth and in the expression of other factors that affect the host-pathogen interaction.

Infect Immun, 1994 Mar, 62(3), 1109 - 17
Sequence determination and mutational analysis of the lly locus of Legionella pneumophila; Wintermeyer E et al.; The lly (legiolysin) locus codes for a 39-kDa protein which confers hemolysis, pigment production, and fluorescence on recombinant Escherichia coli K-12 clones carrying the lly gene . The nucleotide sequences of the lly genes from two Legionella pneumophila isolates were determined . The lly loci exhibited identical nucleotide sequences . They contained open reading frames of 348 amino acid residues, encoding proteins with a deduced molecular mass of 38.9 kDa . N-terminal amino acid sequencing further confirmed that the Lly protein corresponds to the open reading frame sequenced . The amino acid sequence of the Lly protein exhibits a high degree of homology with the sequences of the MelA protein responsible for melanin production in the freshwater bacterium Shewanella colwelliana and the 4-hydroxyphenylpyruvate dioxygenase of Pseudomonas spp . 4-Hydroxyphenylpyruvate dioxygenase is involved in the degradation of aromatic amino acids in various organisms . An Lly-negative mutant of L . pneumophila Philadelphia I derivative JR32 and an Lly-positive transcomplementant were constructed . The Lly-negative mutant lost the ability to produce brown pigment and to confer fluorescence but retained hemolysis . Introduction of a plasmid carrying the lly locus restored pigment production and fluorescence . Intracellular survival of L . pneumophila in U937 macrophage-like cells and in Acanthamoeba castellanii was not affected by mutagenization of the lly locus.

Mol Biochem Parasitol, 1994 Mar, 64(1), 111 - 20
Molecular cloning of ras and rap genes from Entamoeba histolytica; Shen PS et al.; To better understand growth regulation in the protozoan parasite Entamoeba histolytica, ameba genes homologous to the ras oncogene and rap (Krev-1) anti-oncogene were cloned . Two putative ameba ras genes (Ehras1 and Ehras2) were identified, which contain 205 and 203 amino acid (aa) open reading frames (ORFs), respectively . The Ehras1 ORF shows an 91% positional identity with that of Ehras2, a 55% identity with Dictyostelium discoideum (Dd) ras, and a 47% identity with human (Hs) ras . Two ameba rap genes (Ehrap1 and Ehrap2) were identified, both of which contain 184-aa ORFs . The Ehrap1 ORF shows a 93% positional identity with that of Ehrap2, a 60% identity with Dd rap, a 61% identity with Hs Krev-1, and a 45% identity with that of Ehras1 . Conserved aa in each ameba ras and rap ORF include GTP-binding sites, effector site, site of ADP-ribosylation by Pseudomonas exoenzyme S, and COOH-terminus CAAX . As all Xs = Leu or Phe, ameba ras and rap proteins may be gerenylgerenylated and not farnesylated . Both ras and rap genes are transcribed by trophozoites . A single 21-kDa ameba ras protein reacts with the rat Y13-259 anti-ras monoclonal antibody, which is located on the cytosolic side of the plasma membrane . These are the first ras and rap genes identified from a protozoan parasite.

Leuk Lymphoma, 1994 Mar, 13(1-2), 1 - 10
Recombinant single-chain immunotoxins against T and B cell leukemias; Kreitman RJ et al.; Interleukin 2 (IL2) receptors (IL2R's) are found on malignant cells in many human leukemias and lymphomas and are expressed by activated T cells in many autoimmune disorders . Anti-Tac(Fv), a single-chain protein composed of the variable heavy and light domains of the anti-IL2R monoclonal antibody anti-Tac, can be genetically fused to derivatives of Pseudomonas exotoxin (PE) or diphtheria toxin (DT) to form potent immunotoxins . We have shown that anti-Tac(Fv) binds to low affinity IL2R's on fresh chronic lymphocytic leukemia (CLL) and adult T-cell leukemia (ATL) cells and can target either toxin to kill those cells . Anti-Tac(Fv)-PE40, containing the truncated form of PE without its binding domain, was cytotoxic to malignant cells from 8 of 8 ATL patients tested, with IC50's ranging from 0.11 to 5.5 ng/ml . Anti-Tac(Fv)-PE40KDEL, a derivative of anti-Tac(Fv)-PE40 which contains the KDEL carboxyl terminus, was more cytotoxic toward cells from all ATL patients and also killed CLL cells from 8 of 16 patients . DT388-anti-Tac(Fv), containing amino acids 1-388 of DT fused to the amino terminus of anti-Tac(Fv), was less cytotoxic than anti-Tac(Fv)-PE40 on ATL cells from 4 of 5 patients, but was cytotoxic toward CLL cells from 12 of 16 patients . DT388-IL2, where IL2 is substituted for anti-Tac(Fv), is similar to DAB389IL2, an IL2-toxin currently in clinical trials . DT388-IL2 and DAB389IL2 differ by only a few amino acids and have equal cytotoxic activity . DT388-IL2 was cytotoxic toward ATL cells from all patients tested, but usually required much higher concentrations than anti-Tac(Fv)-PE40 and was poorly active against CLL cells . Thus, recombinant toxins containing anti-Tac(Fv) are cytotoxic toward freshly isolated CLL and ATL cells and will be studied further as potential therapy for IL2R-related disorders.

J Clin Pharmacol, 1994 Mar, 34(3), 255 - 9
Absolute bioavailability and absorption characteristics of aerosolized tobramycin in adults with cystic fibrosis; Cooney GF et al.; Administration of antibiotics by the inhalational route has become part of standard protocols for treatment of and prophylaxis for Pseudomonal pneumonias in patients with cystic fibrosis . For tobramycin, however, limited data are available on the aerosol absorption patterns, and no absolute bioavailability data for tobramycin exist . The purpose of this study was to measure the absolute bioavailability and systemic absorption characteristics of tobramycin when administered in high doses by a nebulizer . Multiple serum concentrations of tobramycin were measured after administration of an intravenous dose (mean, 2.9 mg/kg every 6 hours) and after an inhalational dose (5.6 mg/kg over 1 hour) . Inhalational doses were superimposed over the "tail" of a steady-state intravenous dose to improve the sensitivity of the assay procedure (Abbott-TDX) . Absolute bioavailability (F) was determined from AUC ratios normalized for dose . Model-independent pharmacokinetic parameters (volume of distribution {Vss} and total clearance {CLt}) were determined for each subject . Absorption characteristics (absorption rate constant {Ka} and mean absorption time {MAT}) were assessed after calculation of the cumulative fraction of drug absorbed, amount of bioavailable drug, and percent remaining to be absorbed per unit time using the Loo-Riegelman method . Three men and three women completed the study, and all received concurrent doses of ceftazidime . Mean absolute bioavailability (+/- standard deviation) was 9.13% (+/- 3.82), and the rate of absorption into the systemic circulation was consistent with a zero-order model profile for all subjects . Mean absorption time values reflected a wide degree of subject variability and ranged from approximately 15 to 150 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Microbiology, 1994 Mar, 140 ( Pt 3), 509 - 16
Cloning of a second non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762: sequence analysis, expression in Streptomyces lividans and enzyme purification; Pelletier I et al.; The gene for BPO-A1, one of two non-haem bromoperoxidases in the tetracycline and 7-chlorotetracycline producer Streptomyces aureofaciens ATCC 10762, was cloned in the positive selection vector pIJ699 and expressed in Streptomyces lividans TK64 . The cloned bromoperoxidase was over-produced up to 2800-fold by the S . lividans TK64 transformant . By taking advantage of the over-production of BPO-A1 and the heat stability of the enzyme, a new and simple purification procedure was developed . Subcloning into the vector pIJ487 and screening of recombinants by a newly developed histochemical assay located the bpoA1 gene on a 2.1 kb BamHI-HindIII fragment . The nucleotide sequence of the 2.1 kb fragment was determined; the bpoA1 gene was identified within the sequence on the basis of the biased codon usage of Streptomyces genes and the presence of a nucleotide sequence encoding the N-terminal amino acid sequence obtained from the purified BPO-A1 . Comparison of the deduced primary structure of BPO-A1 with those deduced for the non-haem chloroperoxidase CPO-P from Pseudomonas pyrrocinia and the bromoperoxidase BPO-A2 from S . aureofaciens ATCC 10762 gave amino acid sequence identities of 49% and 40%, respectively.

Microbiology, 1994 Mar, 140 ( Pt 3), 499 - 508
The lower pathway operon for benzoate catabolism in biphenyl-utilizing Pseudomonas sp . strain IC and the nucleotide sequence of the bphE gene for catechol 2,3-dioxygenase; Carrington B et al.; Pseudomonas sp . strain IC is able to grow on biphenyl, 3-methylbiphenyl and 4-methylbiphenyl . These are converted to benzoate and the corresponding methylbenzoates . The lower pathway genes for the catabolism of the benzoates were cloned on a 22 kb HindIII fragment . Hybridization with gene-specific probes from the meta pathways of other catabolic plasmids showed that the gene order was identical to that of the operons carrying the same function from TOL plasmids . The nucleotide sequence of a 1241 bp region carrying the whole of the bphE gene (for a catechol 2,3-dioxygenase) and the 5' end of the downstream bphG gene (for 2-hydroxy semialdehyde dehydrogenase) was determined . Both genes showed a high degree of homology with genes encoding isofunctional proteins from other Pseudomonas strains . The upper pathway genes for the conversion of biphenyl to benzoate have also been cloned but no linkage with the lower pathway operon has been detected . Pseudomonas strain IC contains a large plasmid pWW110 (> 200 kb) and there are indications that this plasmid carries the bph genes.

Southeast Asian J Trop Med Public Health, 1994 Mar, 25(1), 144 - 51
Effects of tunicamycin on the pH-activity pattern of acid phosphatase in Pseudomonas pseudomallei; Kondo E et al.; The liquid culture of Pseudomonas pseudomallei shows a complex feature in in the pH-activity pattern of acid phosphatase, not a single peak curve . There was an evident tendency that the higher activity shifted to the higher pH range with the growth of culture . The culture in the presence of tunicamycin (20 micrograms/ml) showed a decreased activity selectively in the higher pH range, while the activity in the lower pH was more heat-labile . The bacterial cells grown on agar plates containing tunicamycin were more heat-labile than the untreated control cells . The glucosidase-treatment reduced the enzymatic activity (of the phosphatase-active fractions from the living cells) with the shift of the optimum pH to lower pH . These observations together with some collateral findings suggest that the pH-activity pattern of acid phosphatase in P . pseudomallei is associated with the development of precursor enzyme proteins to mature glycoproteins.

Plasmid, 1994 Mar, 31(2), 138 - 47
Characteristics of IS401, a new member of the IS3 family implicated in plasmid rearrangements in Pseudomonas cepacia; Byrne AM et al.; We have determined the nucleotide sequence of IS401, an insertion sequence implicated in rearrangements of a 170-kb cryptic plasmid from Pseudomonas cepacia . Our analysis focused on a 4066-bp plasmid fragment containing adjacent copies of IS401 and of IS408, an element reported previously to activate gene expression in P . cepacia . One objective was to determine if an apparent increase in the copy number of IS401 in strains carrying adjacent plasmid copies of these two elements might be due to readthrough transcription of an IS401 transposase gene from an outwardly directed promoter within IS408 . This possibility was ruled out by nucleotide sequence analysis of the 4066-bp plasmid fragment, which indicated that the major open reading frames of IS401 were oriented in the direction of IS408 . IS401 was 1316 bp in length and had 26-bp terminal inverted repeats flanked by 3-bp direct duplications of adjacent DNA . It was closely related to the IS3 family elements IS51 from P . savastanoi and IS3411 from Escherichia coli . Pertinent features of IS408 are also discussed.

Chest, 1994 Mar, 105(3), 837 - 41
Single lung transplantation in patients with systemic disease; Levine SM et al.; OBJECTIVE: To report functional results and survival in patients undergoing single lung transplantation (SLT) for pulmonary involvement associated with systemic disease or prior malignancy, criteria traditionally considered contraindications to SLT . DESIGN: Case series . SETTING: The University of Texas Health Science Center at San Antonio . PATIENTS: Nine patients who have undergone SLT for end-stage lung disease: four patients with sarcoidosis; two patients with limited scleroderma; and three patients with prior malignancies (two with prior lymphoma and bleomycin-induced pulmonary fibrosis and one who received two bone marrow transplants for acute lymphocytic leukemia and subsequently developed chemotherapy-induced pulmonary fibrosis) . MEASUREMENTS: Pulmonary function testing, exercise oximetry, quantitative ventilation-perfusion lung scanning . Actuarial survival . RESULTS: All patients had marked improvement in pulmonary function, exercise oximetry, and quantitative ventilation perfusion to the SLT . One patient with scleroderma died 90 days postoperatively from Pseudomonas pneumonia with a sepsis syndrome . One patient with sarcoidosis died 150 days postoperatively from disseminated aspergillosis . At autopsy, there was no evidence of recurrent fibrosis or sarcoidosis in the transplanted lungs in either of these two patients . The seven surviving patients have returned to work or school and are conducting all activities of daily living without pulmonary disability . The 1- and 2-year actuarial survival rates in these nine patients is 68.6 percent as compared with the 1- and 2-year actuarial survival rates of 66.3 percent and 55.8 percent in the remainder of our SLT group as a whole (n = 49) . Despite pharmacologic immunosuppression, there is no evidence of recurrent malignancy in the 3 patients with prior malignancies . CONCLUSIONS: We conclude that carefully selected patients with end-stage lung involvement related to systemic disease or chemotherapy-induced fibrosis may benefit from SLT.

Biochem Biophys Res Commun, 1994 Feb 28, 199(1), 41 - 5
Alcoholysis of epsilon-decalactone with polyethylene glycol-modified lipase in 1,1,1-trichloroethane; Furukawa M et al.; Lipase from Pseudomonas cepacia was modified with 2,4-bis{O-methoxypoly(ethylene glycol)}-6-chloro-s-triazine, activated PEG2, to form PEG-lipase . The PEG-lipase is soluble and active in organic solvents . It catalyzes alcoholysis of racemic epsilon-decalactone with ethanol in 1,1,1-trichloroethane to form (R)-hydroxydecanoic acid ethyl ester . No alcoholysis of (S)-decalactone takes place . These results were discussed in relation to carbon number of n-alcohol, optimum temperature and comparison with modified and non-modified lipases.

J Mol Biol, 1994 Feb 25, 236(3), 759 - 85
High resolution structures of holo and apo formate dehydrogenase; Lamzin VS et al.; Three-dimensional crystal structures of holo (ternary complex enzyme-NAD-azide) and apo NAD-dependent dimeric formate dehydrogenase (FDH) from the methylotrophic bacterium Pseudomonas sp . 101 have been refined to R factors of 11.7% and 14.8% at 2.05 and 1.80 A resolution, respectively . The estimated root-mean-square error in atomic co-ordinates is 0.11 A for holo and 0.18 A for apo . X-ray data were collected from single crystals using an imaging plate scanner and synchrotron radiation . In both crystal forms there is a dimer in the asymmetric unit . Both structures show essentially 2-fold molecular symmetry . NAD binding causes movement of the catalytic domain and ordering of the C terminus, where a new helix appears . This completes formation of the enzyme active centre in holo FDH . NAD is bound in the cleft separating the domains and mainly interacts with residues from the co-enzyme binding domain . In apo FDH these residues are held in essentially the same conformation by water molecules occupying the NAD binding region . An azide molecule is located near the point of catalysis, the C4 atom of the nicotinamide moiety of NAD, and overlaps with the proposed formate binding site . There is an extensive channel running from the active site to the protein surface and this is supposed to be used by substrate to reach the active centre after NAD has already bound . The structure of the active site and a hypothetical catalytic mechanism are discussed . Sequence homology of FDH with other NAD-dependent formate dehydrogenases and some D-specific dehydrogenases is discussed on the basis of the FDH three-dimensional structure.

J Biol Chem, 1994 Feb 18, 269(7), 5346 - 57
The substrate specificity of Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase . Polar probes of the enzyme's myristoyl-CoA recognition site; Lu T et al.; Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is a monomeric enzyme that is essential for vegetative growth . Nmt1p catalyzes the co-translational transfer of myristate from CoA to the amino-terminal Gly of cellular proteins in an ordered Bi Bi reaction mechanism that initially involves binding of myristoyl-CoA to the apoenzyme . Forty one fatty acid analogs were synthesized to define features in the acyl chain of myristoyl-CoA which are important determinants of its recognition by Nmt1p's acyl-CoA binding site as well as to help us deduce the structure of the binding site itself . These analogs included dicarboxylic acids, omega-nitrocarboxylic acids, analogs equivalent in length to C13:0-C15:0 which contain electronegative halogens at their omega-termini, hydroxytetradecanoic acids with hydrogen replaced by OH from C3 to C13, and azidophenyl-containing fatty acids with the linear azide unit attached either meta or para to phenyl and with variations in the length of their methylene chains . These compounds were converted to their CoA derivatives using Pseudomonas acyl-CoA synthetase and then surveyed as substrates for purified Nmt1p in an in vitro assay system that included an octapeptide derived from residues 1-8 of the human immunodeficiency virus Pr55gag polyprotein precursor . The results suggest that the myristoyl-CoA binding site contains a conical-shaped "receptor" that interacts with the omega-terminus of the bound acyl chain of acyl-CoAs . The acuteness of this cone determines the enzyme's capacity to accommodate steric bulk at the omega-terminus as well as Nmt1p's sensitivity to the distance between the eclipsed C5-C6 bond of a bound acyl chain and its omega-terminus . The activity profile of the various analog-CoAs also indicates that the enzyme's myristoyl-CoA binding site can accommodate fatty acid analogs with marked increases in polarity at their omega-terminus (compared to C14:0) as long as their chain length is equivalent to that of myristate.

Cancer Res, 1994 Feb 15, 54(4), 1059 - 64
Cytotoxicity of recombinant Fab and Fv immunotoxins on adult T-cell leukemia lymph node and blood cells in the presence of soluble interleukin-2 receptor; Saito T et al.; Single-chain immunotoxins anti-Tac(Fv)-PE40 and anti-Tac(Fv)-PE40KDEL, composed of variable domains of the anti-Tac monoclonal antibody and truncated forms of Pseudomonas exotoxin, have shown potent cytotoxic activity against malignant peripheral blood mononuclear cells (PBMCs) from adult T-cell leukemia (ATL) patients originating from the Caribbean . However, several clinically important issues have not previously been addressed . These include the potential of soluble interleukin 2 receptor in ATL patients to block immunotoxin effectiveness, the relative sensitivity of malignant lymph node cells (LNCs) versus PBMCs, the effect of an immunotoxin with a prolonged half-life, and finally whether ATL cells from patients in Japan have toxin sensitivity equal to those of the Caribbean patients . To resolve these questions, we studied 32 malignant PBMC and LNC samples from 30 ATL patients from Japan . PBMCs from 27 of 27 patients were very sensitive with 50% inhibition of protein synthesis achieved with 0.02-0.85 ng/ml (0.3-13 pM) of anti-Tac(Fv)-PE40KDEL or anti-Tac(Fv)-PE40 . LNCs had sensitivity very similar to that of PBMCs in the five patients tested . The fully recombinant immunotoxin, anti-Tac(Fab)-PE40, which has 8-10 times the t1/2 alpha and beta compared to the Fv-immunotoxins, was also very cytotoxic toward cells from 27 of 27 patients tested with 50% inhibition of protein synthesis of 0.08-25 ng/ml . It was found that purified soluble interleukin 2 receptor added to the cytotoxicity assay decreased the cytotoxic activity of anti-Tac(Fv)-PE40KDEL or anti-Tac(Fab)-PE40, but that 1 x 10(4) units/ml or less had minimal competitive effects . It was found that ATL patients who have responded even incompletely to conventional chemotherapy have soluble interleukin 2 receptor levels lower than this at posttreatment . We conclude that recombinant immunotoxins containing anti-Tac(Fv) are effective against Japanese ATL PBMCs or LNCs and might be most effective if used in vivo after conventional chemotherapy . If it is found in humans that the effectiveness of single-chain recombinant toxins is limited by short half-life, anti-Tac(Fab)-PE40 should be considered as an alternative agent.

Cancer Res, 1994 Feb 15, 54(4), 1008 - 15
Transforming growth factor-alpha-Pseudomonas exotoxin fusion protein (TGF-alpha-PE38) treatment of subcutaneous and intracranial human glioma and medulloblastoma xenografts in athymic mice; Phillips PC et al.; Epidermal growth factor receptor (EGFR) is amplified or overexpressed in many malignant gliomas and other primary brain tumors but is low or undetectable in normal brain . In the present study, this differential expression has been exploited for targeted brain tumor therapy using a TGF-alpha-Pseudomonas exotoxin recombinant toxin, TGF-alpha-PE38 . In vitro experiments demonstrate that the cytotoxicity of this fusion protein is primarily determined by tumor EGFR expression and that TGF-alpha-PE38 cytotoxicity is abolished by pretreatment with excess epidermal growth factor . Treatment with i.p . TGF-alpha-PE38 in nude mice bearing glioblastoma or medulloblastoma s.c . xenografts produced tumor regression and growth delay . For intracranial xenograft implants treated with i.p . TGF-alpha-PE38, significant increases in median survival were noted only for tumors with the highest EGFR expression . However, intracranial tumors treated with a single intratumoral injection of TGF-alpha-PE38 showed increased survival in all xenografts tested . These results indicate that TGF-alpha-PE38 is active against primary human brain tumors ranging from moderate to high EGFR expression . For intracranial tumors, however, the higher survival rates produced by intracranial injection of TGF-alpha-PE38 than by continuous i.p . administration suggest that increased drug clearance or impaired drug delivery reduces the efficacy of systemic TGF-alpha-PE38 . Direct delivery of TGF-alpha-PE38 into brain tumors by controlled-release biodegradable polymers or intratumoral implanted catheters, or intrathecal administration into the colony stimulating factor of patients with leptomeningeal metastasis, may represent clinically useful applications of recombinant toxin therapy in tumors with high EGFR expression.

J Mol Biol, 1994 Feb 11, 236(1), 377 - 8
Crystallization of catechol-1,2 dioxygenase from Pseudomonas arvilla C-1; Earhart CA et al.; The metalloenzyme catechol 1,2-dioxygenase from Pseudomonas arvilla C-1 consists of three isozymes formed by combinations of two non-identical subunits; alpha alpha, alpha beta and beta beta; with molecular masses of 59,000, 63,000 and 67,000 Da, respectively . The alpha alpha isozyme crystallizes in the orthorhombic space group C222(1) with unit cell dimensions a = 62.7 A, b = 71.5 A, c = 187.1 A . The rectangular plates diffract to 2.6 A resolution . This is the first dioxygenase to be crystallized that uses catechol as a substrate . Comparison of the structure of this enzyme with protocatechuate 3,4-dioxygenase will provide basic information about the mechanisms of subunit association, substrate selectivity, and the origins of metabolic diversity in enzymes.

Am J Pediatr Hematol Oncol, 1994 Feb, 16(1), 22 - 6
Bone marrow transplantation for sickle cell disease . The United States experience; Johnson FL et al.; PURPOSE: As of June 1992, five patients with sickle cell disease had been treated by matched sibling bone marrow transplantation in the United States . PATIENTS AND METHODS: Three patients underwent transplantations for complications related to sickle cell disease, two with previous cerebrovascular accidents (CVAs) and one who had had multiple severe vasoocclusive crises . Two patients had other indications for allogeneic bone marrow transplantation: one had acute myeloid leukemia and the other had Morquio's disease . The patients' ages ranged from 3 to 10 years, and four were girls . Ages of the donors ranged from 4 to 13 years; four of the donors were boys and three carried the sickle cell trait . For four patients, the preparative regimen consisted of busulfan and cyclophosphamide given either alone or combined with antithymocyte globulin (ATG) . The patient with leukemia was prepared with cyclophosphamide and total body irradiation (TBI) . The regimens for prophylaxis of graft-versus-host disease (GVHD) included various combinations of cyclosporine A, methotrexate, and prednisone . RESULTS: The patient with Morquio's disease failed to engraft but underwent a successful retransplantation from the same donor . All patients eventually demonstrated donor engraftment and the donor's hemoglobin electrophoretic pattern posttransplant . Two patients had moderately severe GVHD of the skin and gastrointestinal tract, which resolved with prednisone therapy . One of these patients developed transient chronic GVHD involving the skin . Other acute complications included mild venoocclusive disease of the liver, central line infection with bacteremias, uterine hemorrhage in one patient, and pseudomonas sepsis in another . CONCLUSIONS: Both patients who underwent transplantation after CVAs have experienced subsequent neurological events . However, with a median follow-up of 16 months (range 8 months to 9.3 years), all patients are surviving in good to excellent clinical condition and appear to have benefitted from treatment by bone marrow transplantation.

Am Fam Physician, 1994 Feb 1, 49(2), 427 - 31
Malignant external otitis: a case report and review; Evans P et al.; Malignant external otitis is an unusual but serious and potentially fatal condition that has only recently been described . It is an invasive pseudomonal infection of the external auditory canal and deep periauricular tissues that characteristically involves the bone and adjacent cartilaginous structures, and it may lead to osteomyelitis of the base of the skull . It typically occurs in elderly diabetic patients . Malignant external otitis can cause severe pain, necrosis of the external auditory canal and progressive palsies of the facial and cranial nerves . Treatment consists of debridement of external auditory canal granulation tissue and long-term therapy with an antipseudomonal cephalosporin or an antipseudomonal penicillin plus an aminoglycoside.

Mol Microbiol, 1994 Feb, 11(3), 489 - 500
VsrA, a second two-component sensor regulating virulence genes of Pseudomonas solanacearum; Schell MA et al.; The wilt-inducing phytopathogen Pseudomonas solanacearum produces several extracellular virulence factors, both polysaccharides (EPS I) and proteins (EXPs), which are independently regulated by a LysR-type transcriptional regulator, PhcA, and a histidine kinase sensor, VsrB . Here we characterize a third locus, vsrA, which is also required for normal production of EPS I, some EXPs and wilt disease . Analysis of eps::lacZ reporters in vsrA mutants showed that, like vsrB and phcA, vsrA is required for maximal expression (transcription) of eps, which contains some of the genes necessary for production of EPS I . Unlike vsrB and phcA mutants, however, eps transcription (and EPS I production) by vsrA mutants varies from 3 to 17% of wild-type levels, depending on growth conditions . Inactivation of vsrA also causes a dramatic reduction in production of three species of EXPs (28 kDa, 48 kDa, and 66 kDa), and an apparent increase in production of a few other EXPs . Unlike most other EPS-deficient P . solanacearum strains, vsrA mutants caused almost no disease symptoms when 10(4) cells were stem-inoculated into tomato plants . This correlated with a greater than 10-fold reduction in their ability to grow in planta . vsrA was cloned from a P . solanacearum genomic library by complementation of the vsrA mutant and was further subcloned on a 2.3 kb DNA fragment . PhoA fusion analysis and subcellular localization of the vsrA gene product in Escherichia coli maxicells suggest that it is a 53 kDa membrane-associated protein . Analysis of the nucleotide sequence of vsrA revealed a 502 residue open reading frame with homology to the histidine kinase domain of sensors in the two-component regulator family . This discovery shows that EPS I production by P . solanacearum is simultaneously controlled by dual two-component sensors.

Arch Biochem Biophys, 1994 Feb 1, 308(2), 400 - 6
New subunit in L-phenylalanine oxidase from Pseudomonas sp . P-501 and the primary structure; Mukouyama EB et al.; L-phenylalanine oxidase from Pseudomonas sp . P-501 was shown by isoelectric focusing and HPLC experiments to consist of two kinds of nonidentical subunits . The newly identified subunit is designated as alpha, and the larger subunit, which has been reported previously, as beta . The apparent molecular weight of alpha subunit was estimated to be 8200 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . From the molecular mass of each subunits and their contents in the enzyme, the enzyme was shown to be composed to two alpha and two beta subunits . The amino acid sequence analysis of alpha subunit showed that the subunit consists of 92 amino acid residues and contains considerably hydrophobic arrangements and the candidate for the common sequence characteristic of the AMP binding in the FAD binding domain.

J Bacteriol, 1994 Feb, 176(4), 1025 - 36
Identification of a putative alternate sigma factor and characterization of a multicomponent regulatory cascade controlling the expression of Pseudomonas syringae pv . syringae Pss61 hrp and hrmA genes; Xiao Y et al.; The Pseudomonas syringae hrp and hrmA genes controlling pathogenicity and elicitation of the hypersensitive response and the avr genes controlling host range have been shown previously to be regulated by carbon, nitrogen, pH, osmolarity, and hypothetical plant factors . In P . syringae pv . syringae Pss61, inactivation of hrp complementation groups II and XIII reduced expression of a plasmid-borne hrmA'-lacZ fusion . The hrp regions II and XIII were cloned on separate plasmids and shown to enhance the activity of the hrmA promoter in Escherichia coli MC4100 transformants at least 100-fold . The nucleotide sequence of region XIII revealed two open reading frames (hrpR and hrpS) whose deduced products share homology with P . syringae pv . phaseolicola NPS3121 HrpS and are both related to the NtrC family of two-component signal transduction systems . HrpR and HrpS differ from most members of the protein family by lacking an amino-terminal domain which modulates the regulatory activity . A single open reading frame, hrpL, whose product shares homology with AlgU, a putative alternate sigma factor of P . aeruginosa, as well as with the related alternate sigma factors was identified within region II . Key domains are partially conserved . Inactivation of hrpS in Pss61 repressed expression of a plasmid-borne hrpL'-lacZ fusion carried by pYXPL1R, and transformation of MC4100(pYXPL1R) with a plasmid carrying hrpRS increased hrpL promoter activity at least 200-fold . Neither hrpS nor hrpR, when cloned on separate plasmids, activated the hrpL promoter activity individually . The expression of hrpL when directed by a lac promoter was sufficient to express a set of plasmid-borne hrmA'-, hrpJ'-, and hrpZ'-lacZ fusions independently of other hrp genes . The results indicate that hrpRS and hrpL are part of a regulatory cascade in which HrpR and HrpS activate expression of hrpL and HrpL, a putative sigma factor, induces expression of HrpL-responsive genes.

Res Microbiol, 1994 Feb, 145(2), 121 - 7
Cloning and expression of the mercury resistance genes of marine Pseudomonas sp . strain MR1 plasmid pMR1 in Escherichia coli; Rani DB et al.; The mercury resistance determinant of marine Pseudomonas sp . strain MR1 plasmid pMR1 was cloned into a narrow-host-range vector pUC18 . A direct selection of mercury resistant clones was successful and 12 clones were evolved; 9 from direct selection on mercury agar plates and 3 from ampicillin-resistant white colonies . All the predicted clones efficiently volatilized mercury . One of the hybrid plasmids pMRD5, containing a 15.5-kb insert, conferred inducible resistance to both HgCl2 and phenyl mercury acetate with over a 40-fold increase in mer resistance in Escherichia coli HB101 . No DNA homology existed between the mer operon of Pseudomonas sp . strain MR1 and the characterized determinants of Tn501 mer DNA.

Bull Acad Natl Med, 1994 Feb, 178(2), 249 - 57; discussion 258-62
{Therapeutic hopes in excretory azoospermia and genetic risks in congenital aplasia of the vas deferens}; Cognat M et al.; Therapeutical solutions now can be proposed to some excretory azoospermia, using in vitro-fertilization with epididymal sperm . The encouraging results obtained with this new approach should be analyzed with the genetic risk, sometimes encountered in the specific form of azoospermia due to the congenital absence of the vas deferens . This abnormality is to day supposed to represent a moderate form of cystic fibrosis (CF), corresponding to a genital phenotype of this disease . This suggestion has been firstly induced by similar anatomical findings in the male individuals presenting a classical form of CF . The hypothesis has mainly been confirmed by the real progress in the genetic analysis of this disease . So an abnormally high percentage of known mutations of CF has been demonstrated in the patients with congenital absence of vas deferens . Other arguments such as positive sweat chloride tests, high percentage of sinusitis or presence of anti-pseudomonas antibodies, reinforce this hypothesis . It is the reason why a clinical and biological check-up, prior to any decision of therapy for infertility, in this specific indication should be done in order to propose a genetic counselling to the couple.

Mol Cell Probes, 1994 Feb, 8(1), 1 - 9
Construction of a specific DNA probe for diagnosis of melioidosis and use as an epidemiological marker of Pseudomonas pseudomallei; Sermswan RW et al.; Pseudomonas pseudomallei is a causative agent of melioidosis . The disease manifestations range from fulminant sepsis to asymptomatic seroconversion . In septicemic cases, a mortality rate of 80-90% is reported . Rapid and specific diagnosis has become important to the clinical microbiology laboratory . We have developed a P . pseudomallei-specific DNA probe . The cloned fragment, herein designated pKKU-S23L, contained 1.5 kb of P . pseudomallei chromosomal DNA . A radioactively labelled pKKU-S23L insert could detect 1.5 ng of its genomic DNA or 40,000 P . pseudomallei cells . The probe was highly specific for P . pseudomallei DNA and did not cross-hybridize with DNAs prepared from other related bacteria . Using pKKU-S23L as a probe in total cellular DNA digestions and Southern blot hybridization, we were able to classify 60 P . pseudomallei clinical isolates obtained from individual melioidosis patients into eight categories . Therefore, this probe has a potential not only for use in development of specific detection of bacterial DNA in clinical specimens but also for application in epidemiological studies of P . pseudomallei.

J Appl Bacteriol, 1994 Feb, 76(2), 142 - 8
Physiological aspects of disinfection resistance in Pseudomonas cepacia; Pyle BH et al.; A Pseudomonas cepacia population was isolated which had reduced susceptibility to iodine and maintained resistance when subcultured several times in phosphate buffer . This population was also resistant to iodine after growth in a minimal medium containing glycerol but not glucose . Addition of cAMP to glucose-grown cells caused increased resistance to iodine . Iodine-resistant cultures also demonstrated reduced susceptibility to chlorination but not to heat or metals (Cu/Ag) . The results indicate that halogen resistance can be expressed in varying degrees, dependent on the carbon source, and cAMP may promote this expression . Thus, a catabolite repression-like mechanism may cause resistant cultures grown in some media to become more sensitive to halogens.

Am J Physiol, 1994 Feb, 266(2 Pt 1), C360 - 6
Heterologous expression of delta F508 CFTR results in decreased sialylation of membrane glycoconjugates; Dosanjh A et al.; The cystic fibrosis transmembrane conductance regulator (CFTR) is commonly mutated in cystic fibrosis to the delta F508 CFTR . CFTR has been shown to function as a adenosine 3',5'-cyclic monophosphate-dependent Cl- channel at the cell surface, and there is evidence to suggest that CFTR may also have a role in transmembrane Cl- conductance in intracellular membrane compartments . Studies using cells from cystic fibrosis patients or heterologous expression systems have demonstrated that defective Cl- conductance at the cell surface and defective acidification of the Golgi compartment are associated with the presence of mutant forms of CFTR . It is possible that mutation of CFTR could also result in altered Golgi function, consistent with reports of changes in the glycosylation of cell surface and secreted glycoproteins in cystic fibrosis . Glycosylation of cell surface glycoproteins, particularly levels of sialylation, may also be related to the increased binding of Pseudomonas to cystic fibrosis cells . The current study was undertaken to compare the sialylation of cell surface glycoconjugates in heterologous cells overexpressing normal CFTR or delta F508 CFTR . The presence of sialylated residues on cells was assessed by the surface binding of the specific lectins, wheat germ agglutinin and elderberry bark lectin . A fluorescent cholera toxin B subunit probe was used to measure surface binding to sialylated gangliosides in transfected cells . Our studies show that cells lacking CFTR and cells expressing normal CFTR have unaltered levels of sialylation . In contrast, cells expressing the delta F508 CFTR have significantly decreased amounts of sialylated glycoproteins and gangliosides on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1994 Jan 28, 138(1-2), 27 - 33
Construction of hybrid biphenyl (bph) and toluene (tod) genes for functional analysis of aromatic ring dioxygenases; Hirose J et al.; Multicomponent enzyme complexes of biphenyl (BP) dioxygenase (Dox) encoded by the gene cluster, bphA1A2A3A4 in Pseudomonas pseudoalcaligenes strain KF707 {Taira et al., J . Biol . Chem . 267 (1992) 4844-4858} and toluene Dox encoded by the gene cluster, todC1C2BA in P . putida strain F1 {Zylstra et al., J . Biol . Chem . 264 (1989) 14940-14946}, show high homologies (approx . 60%) for the corresponding subunit component in spite of the fact that they have discrete substrate specificities . We constructed hybrid gene clusters by replacing the gene component(s) between the large and small subunits of terminal Dox in the bph and tod gene clusters, and analyzed the function of a novel hybrid aromatic ring Dox . Escherichia coli cells expressing the hybrid gene clusters, todC1::bphA2A3A4, todC1C2::bphA3A4 and bphA1::todC2::bphA3A4, gained the ability to convert benzene-toluene and their derivatives to the dihydrodiols, indicating that the hybrid terminal Dox composed of TodC1::BphA2 and BphA1::TodC2 forms a functionally active multicomponent Dox associated with ferredoxin (Fer) (BphA3) and Fer reductase (BphA4) . Moreover, hybrid Dox (composed of TodC1::BphA2A3A4 and TodC1C2::BphA3A4) showed a wide substrate specificity rather similar to that of the wild-type toluene Dox (TodC1C2BA) . On the other hand, the hybrid Dox (BphA1::TodC2::BphA3A4) showed oxidative activities for the same compounds, but the rate of oxidation was dependent upon the substrate . These results suggest that (i) the two subunits of terminal Dox are critically involved in the substrate specificity for BP, benzene and their derivatives, and (ii) the electron transport proteins, Fer and Fer reductase, are exchangeable with one another between the BP Dox and toluene Dox complexes.

Gene, 1994 Jan 28, 138(1-2), 119 - 21
Cloning of cmpE, a plasmid-borne catechol 2,3-dioxygenase-encoding gene from the aromatic- and chloroaromatic-degrading Pseudomonas sp . HV3; Yrjala K et al.; Pseudomonas sp . strain HV3 degrades aromatics and chloroaromatics . It harbours a mega-plasmid, designated pSKY4, from which the gene cmpE, encoding a catechol 2,3-dioxygenase (C23O) catalyzing the conversion of catechol to 2-hydroxymuconic semialdehyde, was cloned and sequenced . The deduced amino acid (aa) sequence shows the highest homology, 52%, to the deduced aa sequences of xylE1 and dmpB . The deduced 307-aa sequence of cmpE contains the extradiol ring-cleavage signature in the same position as other 307-aa C23O-encoding genes.

Proc Natl Acad Sci U S A, 1994 Jan 18, 91(2), 664 - 8
Tat-mediated delivery of heterologous proteins into cells; Fawell S et al.; The Tat protein of human immunodeficiency virus 1 (HIV-1) can enter cells efficiently when added exogenously in tissue culture . To assess if Tat can carry other molecules into cells, we chemically cross-linked Tat peptides (residues 1-72 or 37-72) to beta-galactosidase, horseradish peroxidase, RNase A, and domain III of Pseudomonas exotoxin A (PE) and monitored uptake colorimetrically or by cytotoxicity . The Tat chimeras were effective on all cell types tested, with staining showing uptake into all cells in each experiment . In mice, treatment with Tat-beta-galactosidase chimeras resulted in delivery to several tissues, with high levels in heart, liver, and spleen, low-to-moderate levels in lung and skeletal muscle, and little or no activity in kidney and brain . The primary target within these tissues was the cells surrounding the blood vessels, suggesting endothelial cells, Kupffer cells, and/or splenic macrophages . Tat-mediated uptake may allow the therapeutic delivery of macromolecules previously thought to be impermeable to living cells.

Blood, 1994 Jan 15, 83(2), 426 - 34
Recombinant immunotoxins containing anti-Tac(Fv) and derivatives of Pseudomonas exotoxin produce complete regression in mice of an interleukin-2 receptor-expressing human carcinoma; Kreitman RJ et al.; Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin composed of the variable domains of the monoclonal antibody anti-Tac, which binds to the p55 subunit of the interleukin-2 receptor (IL-2R), and a truncated form of Pseudomonas exotoxin (PE), which does not bind to the PE receptor (Chaudhary et al, Nature 339:394, 1989) . Whereas its cytotoxic activity toward autoimmune and malignant target cells has been established, its efficacy in vivo remains unknown . To establish an animal model, we produced ATAC-4 cells by transfecting the gene encoding the low-affinity IL-2R (p55) into A431 epidermoid carcinoma cells . ATAC-4 cells contained low-affinity IL-2Rs (2 x 10(5)/cell) and formed tumors in nude mice . In tissue culture, protein synthesis in ATAC-4 cells was inhibited 50% (IC50) at 0.06 ng/mL (0.9 pmol/L) of anti-Tac(Fv)-PE40 . IC50s for the derivatives anti-Tac(Fv)-PE38, which is missing PE amino acids 365-380, and anti-Tac(Fv)-PE38KDEL, which contains the same deletion plus the KDEL carboxyl terminus, were 0.04 and 0.025 ng/mL, respectively . All the agents produced complete tumor regressions in ATAC-4 tumor-bearing mice and anti-Tac(Fv)-PE38KDEL had significant antitumor activity at 1% of the LD50 . The dose limiting toxicity of anti-Tac(Fv)-PE38KDEL was from hemorrhagic liver necrosis, which was observed at approximately 55% of the LD50.

Carbohydr Res, 1994 Jan 3, 251, 251 - 67
New acyclic analogues of lipid A: synthesis of 4-phosphonoxybutyl and 3-phosphonoxypropyl glycosides of 2-amino-2-deoxy-D-glucose; Eustache J et al.; Several analogues of lipid A have been synthesized, in which the reducing monosaccharide moiety of the parent molecule has been replaced by an acyclic spacer . The new compounds show high endotoxic activity and are able to protect neutropenic mice against pseudomonas infection, two properties characteristic of LPS-like molecules.

Life Sci, 1994, 54(7), 445 - 53
TGF alpha-PE40 inhibits non-small cell lung cancer growth; Draoui M et al.; The ability of a chimeric toxin containing transforming growth factor alpha (TGF alpha) and truncated Pseudomonas exotoxin A to inhibit NSCLC growth was investigated . TGF alpha-PE40 inhibited binding of 125I-EGF to NSCLC cell lines with an IC50 value of 0.5-3 micrograms/ml . Similarly, other forms of the fusion protein, TGF alpha-PE38 and TGF alpha-PE40Asp553, which have active TGF alpha binding domains, inhibited specific 125I-EGF binding to NSCLC cells with IC50 values of 0.1-2 and 0.05-05 microgram/ml respectively . TGF alpha-PE40 inhibited 35S-methionine uptake by NSCLC cells with an ED50 value of 1-30 ng/ml . TGF alpha-PE38, which has one of the two disulfide pairs of PE40, inhibited amino acid uptake with ED50 values of 3-50 ng/ml whereas TGF alpha-PE40Asp553, which lacks ADP ribosylation activity, had an ED50 > 100 ng/ml . TGF alpha-PE40 inhibited colony formation of NSCLC cells with an LD50 value of 0.008-0.1 ng/ml . Similarly, TGF alpha-PE38 inhibited NSCLC colony formation with LD50 values of 0.002-0.1 ng/ml whereas TGF alpha-PE40Asp553 had an LD50 > 10 ng/ml . Also, TGF alpha-PE40 and TGF alpha-PE38 inhibited NSCLC xenograft formation in nude mice whereas TGF alpha-PE40Asp553 was inactive . These data suggest that TGF alpha-PE40 and TGF alpha-PE38 may be useful agents to inactivate NSCLC cells.

J Fam Pract, 1994 Jan, 38(1), 30 - 2
Swimming and grommets; Cohen HA et al.; BACKGROUND . Traditionally, children with tympanostomy ventilating tubes, or grommets, were advised that water should not enter their ears in order to prevent ear infections . This group of children has been considered somewhat handicapped regarding swimming . We conducted a prospective study to determine if there is a relation between suppurative otitis media and surface swimming in children with grommets . METHODS . Forty-two children with tympanostomy ventilating tubes were included in this study . Of the 42 children, 22 were swimmers and 20 were nonswimmers, who served as the control group . The age range was 3 to 12 years, and there was no difference in the age distribution between the groups . Surface swimming was allowed without earplugs or a bathing cap, although it was mandatory to use polymyxin B-neomycin-hydrocortisone eardrops at bedtime on the day of swimming . No diving was allowed . RESULTS . Three of 22 swimmers and 2 of 20 nonswimmers developed otorrhea . In 4 of the 5 children, the otorrhea was followed by an upper respiratory tract infection . In all cases, a bacterial culture revealed Pseudomonas . The ear drainage was easily controlled with local otic treatment in all the patients . CONCLUSIONS . Taking into consideration the possible risks of infection and bearing in mind the value and joy of swimming to children and parents, families should be reassured that surface swimming does not increase the risk of infection in children with tympanostomy tubes.

Mol Gen Genet, 1994 Jan, 242(1), 9 - 16
Nucleotide sequence analysis and potential environmental distribution of a ferric pseudobactin receptor gene of Pseudomonas sp . strain M114; Morris J et al.; The nucleotide sequence of the Pseudomonas sp . strain M114 pbuA gene, encoding the outer membrane receptor for ferric pseudobactin M114, has been determined . The region sequenced spans 2788 bases of plasmid pCUP3, within which the receptor gene had previously been localised . A single open reading frame, potentially encoding 826 amino acids and including a leader peptide of 44 amino acids, is evident and is followed by an inverted repeat segment, which may act as a transcriptional terminator . A 20 bp region of DNA, having significant homology with the E . coli Fur-binding consensus sequence, is located upstream of the open reading frame . PbuA displays characteristics in common with other outer membrane proteins and displays strong homology with the TonB boxes of both E . coli and Pseudomonas receptors . More extensive homologies were found with the PupA receptor of P . putida WCS358 and the FhuE and BtuB receptors of E . coli . It is suggested that areas exhibiting the least homology between these receptors may represent ferric siderophore-specific recognition sites of the PbuA protein . The deduced amino acid sequence of pbuA was compared with that of pupX, encoding the outer membrane receptor for ferric pseudobactin B10, of Pseudomonas sp . strain B 10 . A direct alignment of the two proteins gave an identity score of 92.5% . The distribution of PbuA-like receptors among Pseudomonas isolates was investigated by DNA-DNA hybridisation analysis . The results suggest that a PbuA-like receptor may be widely distributed among Pseudomonas rhizosphere isolates.

Bioconjug Chem, 1994 Jan-Feb, 5(1), 40 - 6
An immunotoxin with increased activity and homogeneity produced by reducing the number of lysine residues in recombinant Pseudomonas exotoxin; Debinski W et al.; Pseudomonas exotoxin A (PE) is a protein composed of 613 amino acids arranged into three major, and one minor, domains . Immunotoxins (ITs) containing PE38, a mutant form of PE which lacks the cell binding domain (Ia, amino acids 1-252) and 16 amino acids from domain Ib (amino acids 365-380), are extremely potent cytotoxic agents which can cause a complete regression of various human carcinomas grown in nude mice . However, these ITs are a mixture of several different chemical forms since the coupling between the antibody and the toxin may occur between either the light or heavy chain of the antibody and one of the four primary amino groups present on the truncated toxin . To modify the toxin with heterobifunctional crosslinking reagents only at specific sites, we replaced lysines 590 and 606 with glutamines and lysine 613 with arginine (PE38QQR) . We also added two different peptide sequences, each containing a lysine residue, at the N-terminus of PE38 . In one of these the sequence is ANLAEEAFK ("Lys" peptide), and in the other, the sequence is LQGTKLMAEE ("NLys" peptide) . The mutant toxins were coupled using a thioether linkage to monoclonal antibody B3 which recognizes an antigen present in large amounts on many human cancers . PE38QQR-containing recombinant toxins can only be linked to an antibody through the N-terminal methionine or the lysine within the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Plant Microbe Interact, 1994 Jan-Feb, 7(1), 140 - 7
Amino acid residues required for the activity of avrD alleles; Yucel I et al.; Certain Pseudomonas syringae pathovars harbor avrD alleles belonging to two different homology classes . The nonfunctional avrD allele of P . s . pv . glycinea is highly homologous to active class II avrD alleles but has five unique amino acid substitutions . Three of these five amino acid changes were shown to be absolutely required for restoration of avrD activity to the P . s . pv . glycinea allele by oligonucleotide site-directed mutagenesis . They were cysteine 19 to arginine, alanine 280 to valine, and leucine 304 to serine . In addition, changing leucine 301 to phenylalanine was required for high activity . However, alteration of the leucine at position 245 of the P . s . pv . glycinea allele to serine, present in the active alleles, did not affect avrD activity . Results from recombinant gene constructs between the nonfunctional P . s . pv . glycinea avrD gene and the functional allele from P . s . pv . phaseolicola identified six other amino acid residues that may form contextual motifs important for AvrD function . These were a four amino acid stretch comprised of glutamate 41, alanine 42, asparagine 43 and arginine 44 in addition to aspartate 243 and phenylalanine 301 . Some divergence is tolerated within the four amino acid motif, but phenylalanine 301 appears to be necessary for highly active class II avrD proteins.

Mol Plant Microbe Interact, 1994 Jan-Feb, 7(1), 131 - 9
Two different classes of avrD alleles occur in pathovars of Pseudomonas syringae; Yucel I et al.; Considerable variation was observed in the occurrence of avirulence gene D (avrD) in different isolates and pathovars of Pseudomonas syringae . Three functional alleles of avrD were cloned and characterized from P . s . pv . phaseolicola and P . s . pv . lachrymans . These avrD genes occurred on indigenous plasmids in both pathovars, like the allele originally cloned from P . s . pv . tomato . P . s . pv . lachrymans was unique in that it carried two different alleles on plasmids of different sizes . These alleles were cloned on 5.6- or 3.8-kb HindIII fragments that are conserved in several other P . syringae pathovars . Surprisingly, the two avrD alleles from P . s . pv . lachrymans were the most divergent of those compared, with only 85% amino acid identity . Allele 1 from P . s . pv . lachrymans was 95% identical to avrD from P . s . pv . tomato but less similar to the other three avrD genes . These two alleles were accordingly called homology class I . The avrD gene from P . s . pv . phaseolicola and allele 2 from P . s . pv . lachrymans were 97 and 98% identical, respectively, at the amino acid level with the nonfunctional P . s . pv . glycinea allele . These three alleles were therefore grouped into homology class II . Comparison of all the avrD alleles permitted the identification of four amino acid substitutions unique to the P . s . pv . glycinea allele at positions 19, 245, 280, and 304.

Mol Plant Microbe Interact, 1994 Jan-Feb, 7(1), 121 - 30
Characterization of a negative regulator of exopolysaccharide production by the plant-pathogenic bacterium Pseudomonas solanacearum; Kao CC et al.; Wild-type strains of the bacterial wilt pathogen Pseudomonas solanacearum exhibit reduced exopolysaccharide production and virulence when transformed with plasmids carrying the epsR locus . To understand the function of epsR, we used mutagenesis and DNA sequencing to identify the gene responsible for the shutoff of exopolysaccharide production . The epsR gene encodes a 236-amino-acid polypeptide that, based on polypeptide sequence homology, has significant similarity to other proteins of the luxR family of environmentally responsive, two-component regulatory systems . When a mutated copy of the epsR gene was marker-exchanged into the wild-type P . solanacearum chromosome, however, we observed no effect on growth in culture or on exopolysaccharide production . This suggests that the EpsR phenotype becomes apparent only via overproduction of the EpsR protein . By means of an antiserum directed against the EpsR protein, we detected the overproduction of EpsR in cell lysates of a strain of P . solanacearum harboring a multicopy plasmid with an active epsR gene but not in one harboring the same plasmid with a mutated epsR gene.

J Wildl Dis, 1994 Jan, 30(1), 107 - 9
Protocalliphora braueri (Diptera: Calliphoridae) induced pathogenesis in a brood of marsh wren (Cistothorus palustris) young; Warren Y; Infestation with blow fly larvae (Protocalliphora (Trypocalliphora) braueri Hendel) was pathogenic to marsh wren (Cistothorus palustris) young . The mechanism of pathogenicity was Pseudomonas spp . infection of subdermal myiasis-induced lesions and subsequent sepsis . Neither internal organ involvement nor muscle destruction was seen on necropsy of fledglings . Multifocal hepatic necrosis was seen histologically and Pseudomonas sp . was isolated from myiasis sites, liver, and peritoneal cavities.

Prikl Biokhim Mikrobiol, 1994 Jan-Feb, 30(1), 55 - 63
{Tn5-mutagenesis of the styrene-degrading strain Pseudomonas sp . Y2 . Analysis of transformation products and DNA-scopy of the mutants obtained}; Iakimov MM et al.; The bacterium Pseudomonas sp . Y2 using styrene as a sole source of carbon and energy was subjected to transposon Tn5(Kmr) mutagenesis . The mutants were divided into three classes by the ability to grow on styrene, 2-phenylethanol, and phenylacetate . It is shown that 2-phenylethanol is not an obligate metabolite of styrene transformation . This allows the conclusion that in Pseudomonas sp . Y2 styrene can be degraded via consistent oxidation of the side chain by two pathways . For the comparison of strains/mutants and determination of genetic markers, DNA-fingerprinting of total DNA was carried out using electrophoresis of restriction fragments and blot-hybridization with different 32P-DNA-probes.

Postgrad Med J, 1994 Jan, 70(819), 47 - 8
Successful treatment of Pseudomonas paucimobilis haemodialysis catheter-related sepsis without catheter removal; Saltissi D et al.; Infection is the most common complication of long-term central venous access lines . We report a case of central venous haemodialysis catheter infection with Pseudomonas paucimobilis, successfully treated by prolonged antibiotic administration without catheter removal . The implications are discussed.

Biometals, 1994 Jan, 7(1), 30 - 40
Silver resistance in Pseudomonas stutzeri; Slawson RM et al.; Silver resistance was studied in a silver-resistant Pseudomonas stutzeri AG259 strain and compared to a silver-sensitive P . stutzeri JM303 strain . Silver resistance was not due to silver complexation to intracellular polyphosphate or the presence of low molecular weight metal-binding protein(s) . Both the silver-resistant and silver-sensitive P . stutzeri strains produced H2S, with the silver-resistant AG259 strain producing lower amounts of H2S than the silver-sensitive JM303 strain . However, intracellular acid-labile sulfide levels were generally higher in the silver-resistant P . stutzeri AG259 strain . Silver resistance may be due to formation of silver-sulfide complexes in the silver-resistant P . stutzeri AG259 strain.

Appl Environ Microbiol, 1994 Jan, 60(1), 235 - 42
Self-mobilization and organization of the genes encoding the toluene metabolic pathway of Pseudomonas mendocina KR1; Wright A et al.; The toluene metabolic pathway of Pseudomonas mendocina KR1 is chromosomally encoded, but the pathway could be transferred by conjugation from strain KR1 to the chromosome of P . aeruginosa or P . putida . Such transconjugants utilized toluene, p-cresol, and p-hydroxybenzaldehyde . However, transconjugants were unable to further transfer toluene genes to other recipients unless Pseudomonas sex factor R68.45 was present in trans . Although the genes encoding the upper pathway for toluene metabolism in P . mendocina KR1 are sufficiently linked to permit their coordinate mobilization, they were found to be encoded in three independently regulated units: one encoding toluene-4-monooxygenase, a second encoding p-cresol methylhydroxylase and p-hydroxybenzaldehyde dehydrogenase, and a third encoding p-hydroxybenzoate hydroxylase . The last two regulatory units were cloned from the chromosome of a P . aeruginosa transconjugant onto a plasmid designated pRO1999 . Analysis of pRO1999 showed that genes encoding p-cresol methylhydroxylase and p-hydroxybenzaldehyde dehydrogenase are organized as an operon; the gene encoding p-hydroxybenzaldehyde dehydrogenase is transcribed first, and this is followed by transcription of the gene encoding p-cresol methylhydroxylase . This operon is regulated by a positively acting regulator . The P . mendocina KR1 gene encoding p-hydroxybenzoate hydroxylase was linked to, but independently regulated from, the genes encoding toluene-4-monooxygenase, p-cresol methylhydroxylase, and p-hydroxybenzaldehyde dehydrogenase.

J Clin Pediatr Dent, 1994 Spring, 18(3), 215 - 7
A dental complication involving Pseudomonas during chemotherapy for acute lymphoblastic leukemia; Cheatham BD et al.; This is a case report of an 11-year-old white female, diagnosed with acute lymphoblastic leukemia, who developed a dental complication involving endodontic failure and Pseudomonas infection during induction chemotherapy . This unusual set of circumstances resulted in alteration of the child's medical management . Although the oral infection, neutropenia and delayed healing response resulted in interruption of the course of chemotherapy, the child did eventually achieve remission . This case does however demonstrate the importance of dental consultation for oncology patients.

J Cancer Res Clin Oncol, 1994, 120(9), 507 - 12
Cytotoxic effect of a fusion protein from transforming growth factor alpha and Pseudomonas exotoxin on rat and human bladder carcinoma cells in vitro; Kameyama S et al.; A protein formed by fusion of transforming growth factor alpha with Pseudomonas exotoxin (TGF alpha-PE40) has been shown to have the ability to kill or inhibit the growth of several carcinoma cell lines . This study was designed to evaluate the in vitro cytotoxic effects of TGF alpha-PE40 on rat and human bladder carcinoma cell lines with different biological potential, and normal rat urothelial cells . The rat cell lines used were D44c, LMC19, and MYU3L, which were established in our laboratory . Human cell lines used were RT4, T24, and 253J . As a normal control, we used the first-passage culture of normal rat bladder urothelium (RU-P1) . We examined the number and affinity of epidermal growth factor receptors (EGFR) in these cells, the ability of TGF alpha-PE40 to bind EGFR, and the cytotoxic effect of TGF alpha-PE40 and PE40 . Rat cell lines, D44c, LMC19, and MYU3L (EGFR = 4.9 x 10(3)-11.4 x 10(3)/cell) had ED50 values (the concentration of TGF alpha-PE40 needed to reduce the viable cell population by 50%) of 180 pM, 540 pM and 6000 pM respectively; for cI (the concentration required to achieve complete inhibition of growth under continuous serum stimulation) TGF alpha-PE40 concentrations of 10(4) pM, 10(4) pM and higher than 10(4) pM respectively were required . Human cell lines, RT4, T24, and 253J (EGFR = 32 x 10(3)-126 x 10(3)/cell) had ED50 values of 20 pM, 66 pM, and 330 pM respectively and T24 showed cI values of 10(3) pM . RU-P1 (EGFR = 92.6 x 10(3)/cell) had the highest ED50 value of 8000 pM . These data indicate that the susceptibility to TGF alpha-PE40 does not always depend on the number of EGFR, that cells having a relatively small number of EGFR respond well to TGF alpha-PE40, and that normal urothelial cells are more resistant to TGF alpha-PE40 than are cancer cells . The differential effect of TGF alpha-PE40 on normal and neoplastic cells provides a rational basis for its use in vivo to control tumor growth.

J Basic Microbiol, 1994, 34(2), 77 - 85
Pseudomonas acidovorans: a bacterium capable of mineralizing 2-chloroaniline; Hinteregger C et al.; Prolonged adaptation of Ca-alginate immobilized cells of Pseudomonas acidovorans CA28 to a mixture of 3-chloroaniline (3-CA)1) and 2-CA and subsequently to 2-CA as sole substrate led to the isolation of another strain, termed CA50 with the additional capability of utilizing 2-CA as sole source of carbon, nitrogen, and energy . Batch-degradation of 190 mg/l of 2-CA at pH 6.1 by this newly isolated strain was achieved within 3 days, at higher concentrations up to 0.6 g/l increasing lag-phases and degradation periods were observed . Except chloride and ammonium no further metabolites were detectable in the medium . Mineralization of 2-CA proceeds via the modified ortho-cleavage pathway as demonstrated by the presence of catechol 1,2-dioxygenase (C120) activity, which is characterized by its substrate specificity and elution behaviour on DEAE-cellulose.

Arch Virol, 1994, 137(1-2), 185 - 90
Bacteriophages from Bombyx mori; Ackermann HW et al.; Preparations of silkworm larvae contained two large phages with contractile tails (Myoviridae) . One phage was active on Pseudomonas paucimobilis . The other, not cultivated, was one of the largest viruses known.

Rev Argent Microbiol, 1994 Jan-Mar, 26(1), 28 - 35
A decalin-consuming bacterial community; Vitale AA et al.; A bacterial community able to degrade cis- and trans-decalin (decahydronaphthalene) in the presence of n-decane could be isolated . It was composed by a couple of Pseudomonas strains (D1 and D2, respectively) . Neither the community nor the isolated strains were able to grow on decalin alone . D1 grew on decalin plus n-decane . Both could grow on n-decyl alcohol, acetate and adipate . From hydrocarbonated substrates it would be generated a metabolic chain which allows the growth of the community members, and then it resulted to be stable . Characteristics of both strains are described.

Arch Microbiol, 1994, 162(1-2), 75 - 9
Control of the pyrimidine biosynthetic pathway in Pseudomonas pseudoalcaligenes; West TP; The five de novo enzyme activities unique to the pyrimidine biosynthetic pathway were found to be present in Pseudomonas pseudoalcaligenes ATCC 17440 . A mutant strain with 31-fold reduced orotate phosphoribosyltransferase (encoded by pyrE) activity was isolated that exhibited a pyrimidine requirement for uracil or cytosine . Uptake of the nucleosides uridine or cytidine by wild-type or mutant cells was not detectable; explaining the inability of the mutant strain to utilize either nucleoside to satisfy its pyrimidine requirement . When the wild-type strain was grown in the presence of uracil, the activities of the five de novo enzymes were depressed . Pyrimidine limitation of the mutant strain led to the increase in aspartate transcarbamoylase and dihydroorotate dehydrogenase activities by more than 3-fold, and dihydroorotase and orotidine 5'-monophosphate decarboxylase activities about 1.5-fold, as compared to growth with excess uracil . It appeared that the syntheses of the de novo enzymes were regulated by pyrimidines . In vitro regulation of aspartate transcarbamoylase activity in P . pseudoalcaligenes ATCC 17440 was investigated using saturating substrate concentrations; transcarbamoylase activity was inhibited by Pi, PPi, uridine ribonucleotides, ADP, ATP, GDP, GTP, CDP, and CTP.

Plasmid, 1994 Jan, 31(1), 21 - 30
Characterization of high-frequency deletions in the iaa-containing plasmid, pIAA2, of Pseudomonas syringae pv . savastanoi; Soby S et al.; The phytopathogenic bacterium Pseudomonas syringae pv . savastanoi causes olive and oleander knot disease . The bacterium induces the formation of tumorous galls by the synthesis and secretion of the plant hormones trans-zeatin riboside and indole-3-acetic acid into host intercellular spaces . An Italian oleander isolate, PB213, has been observed to lose the ability to synthesize IAA at high frequency, thus becoming non-pathogenic . The IAA genes, located on the 72-kb iaa-containing plasmid, pIAA2 were lost mainly due to two classes of deletions: 18 or 22 kb in length . Both classes of deletions had a common endpoint upstream of the IAA genes . The other endpoints were in areas that flanked the insertion sequence element IS51 . The endpoints are in regions of repetitive DNA of at least 271 bp that have been designated a/b.

Mol Plant Microbe Interact, 1994 Jan-Feb, 7(1), 78 - 90
Syringomycin production among strains of Pseudomonas syringae pv . syringae: conservation of the syrB and syrD genes and activation of phytotoxin production by plant signal molecules; Quigley NB et al.; The syrB and syrD genes of Pseudomonas syringae pv . syringae are predicted to encode proteins that function in the synthesis and export of syringomycin, respectively . Using portions of the syr genes as DNA probes, both genes were shown to be conserved as single copies within a 15-kb or smaller DNA region among a broad spectrum of P . s . pv . syringae strains that produce syringomycin or one of its amino acid analogs, syringotoxin and syringostatin . Strains representative of P . viridiflava and six pathovars of P . syringae failed to hybridize with the gene probes, demonstrating that syr sequences are highly specific to P . s . pv . syringae and related nonpathogenic strains . Maximum parsimony analysis of restriction fragment length polymorphism profiles was used to evaluate relatedness among strains within the syrB and syrD gene region . A tree, conveying the smallest number of evolutionary changes among strains, revealed considerable diversity within the syr gene region; subclusters of strains were identified that appear to share specific qualities relevant to the plant-pathogen interaction . Because both the syrB gene and syringomycin production can be induced in response to plant signal molecules, 42 strains containing homologous syr sequences were tested for signal-mediated induction of toxin production . Over 90% of the toxigenic strains produced larger quantities of toxin when the plant signal molecules, arbutin and D-fructose, were added to syringomycin-minimal medium; 13 of the strains produced > or = 10-fold higher toxin levels . Some strains, such as 5D428, produced toxin only in the presence of these signals.(ABSTRACT TRUNCATED AT 250 WORDS)

Ann Oncol, 1994, 5 Suppl 1, 97 - 103
Immunotoxins: is there a clinical value?
Gottstein C, Winkler U, Bohlen H, Diehl V, Engert A.
Drug targeting is an attractive new approach to killing malignant cells, thereby leaving normal tissue unharmed . A decisive breakthrough was the advent of hybridoma technology, making monoclonal antibodies (MoAb) available in limitless supply . To construct reagents with selectivity for certain tumor cells, MoAbs or Fab' fragments were chemically linked to ribosome-damaging toxins derived from plants or bacteria like ricin, abrin, saporin, Pseudomonas exotoxin (PE), and diphtheria toxin (DT) to form immunotoxins, which combined the selectivity of the carrier moiety with the potency of the toxin moiety . The first generation of these immunotoxins showed impressive results in vitro but in most cases disappointing antitumour effects in animals or humans . By contrast, the second generation of immunotoxins, consisting of either A chain immunotoxins with a greatly improved stability in vivo or so-called 'blocked' ricin immunotoxins, have been demonstrated to be extremely effective in several animal models . Preliminary results of the current clinical trials suggest a possible clinical use of immunotoxins in leukemia and lymphoma patients . Genetically engineered fusion toxins have become available, which consist of a growth factor or a cytokine fused to a toxin moiety . In this paper, we will review the features of the three groups of immunotoxins which are most frequently used, i.e., ricin A chain and similar immunotoxins, blocked ricin immunotoxins, and recombinant toxins constructed with Pseudomonas exotoxin or diphtheria toxin.

Chin J Biotechnol, 1994, 10(4), 233 - 9
Construction and expression of high efficiency expressing plasmid of transforming growth factor alpha-Pseudomonas aeruginosa exotoxin fusion protein TGF alpha-PE40; Xu Y et al.; The XbaI/EcoRI cleaved TGF alpha-PE40 gene from plasmid pXY382 was inserted into the same cloning site of the expression vector pCB604 resulting in the plasmid p2X-TP1 . E . coli BL21 (lambda DE3) cells were transformed with the p2X-TP1 and then induced with IPTG . The product expressed was accumulated mainly in the form of inclusion bodies . The expression level was closely related to the cell density, induction temperature and medium but not to the inductor dosage and the induction period within certain range . The expressed amount of the fusion protein TGF alpha-PE40 was about 50 mg/L.

Planta, 1994, 195(2), 175 - 81
Extensin gene expression is induced by mechanical stimuli leading to local cell wall strengthening in Nicotiana plumbaginifolia; Tire C et al.; Nicotiana plumbaginifolia Viv . harbors a single extensin gene, although related hydroxyproline-rich sequences are present in the genome . Northern analysis showed that the gene is highly expressed in roots and to a lesser extent in stems . Expression in leaves is low but mRNA levels are increased upon infection with the incompatible bacterium Pseudomonas syringae . Extensin transcript levels in leaves were slightly enhanced after wounding and salicylic acid treatment . In-situ hybridization experiments showed high accumulation of extensin mRNA in cells which, at certain stages of development, require reinforcement of their cell walls . The cortical cells in stem nodes and roots, which are put under severe mechanical stress by adjacent developing tissues, tend to express the gene to high levels . Immunolocalization of the extensin protein in stems and roots demonstrated a close association of the protein with lignin deposition . Mature tissues contained more extensin than younger tissues . The extensin promoter was fused to the beta-glucuronidase gene.

Acta Biol Hung, 1994, 45(1), 17 - 23
Investigation of chromium (VI) tolerant bacteria; Vincze G et al.; Pseudomonas strains were isolated from a heavy metal contaminated sludge . Some of the strains grew on chromium (VI) containing medium, and others were inhibited in their growth . Cells were able to remove chromium from the medium . This ability was independent of the rate of their growth . The concentration of chromium (VI) of the media decreased with 15-20 ppm in presence of 10(9) cells . The chromium (VI) was converted into a reduced form of chromium by bacteria . The reduced form of chromium was bound in the cell wall . The bound form of chromium could be recovered more or less with different chemicals . The best percentages of recovery were achieved with NaOH solution.

Cell Biophys, 1994, 24-25, 9 - 14
Removal of endotoxin from antibody preparations for clinical use . Assessment of polymyxin-sepharose CNBr affinity chromatography; Lahiri VL et al.; Despite attempts to maintain asepsis, good manufacturing practices, and the use of terminal sterilization by millipore filtration, the nuclear practitioner is always worried about the possibility of endotoxin contamination . Methods, such as ion-exchange chromatography, have been tried for removing endotoxins during the preparation of radiolabeled antibodies, and so on . As suggested by Stevenson (1990), we evaluated the Issekutz technique (1) of endotoxin removal by affinity chromatography using a polymyxin cyanogen bromide (CNBr) Sepharose column . The endotoxin content of millipore filtrates of heat killed/sonicated suspensions of Pseudomonas pyocyaneus, E . coli were measured using a Sigma (St . Louis, MO) Endotoxin Assay Kit before and after filtration through such columns and compared with the results obtained using gel exclusion and ion-exchange columns of the same length and diameter . Reduction of endotoxin content to undetectable levels by the polymyxin column was observed . The use of such columns for terminal endotoxin removal analogous to terminal sterilization is advocated especially when developing a radiopharmaceutical such as radiolabeled antibodies for in house use.

Cell Biophys, 1994, 24-25, 249 - 57
Targeting phosphodiesterases as a strategy for killing tumor cells; Deonarain MP et al.; Ribonucleases (RNases) are being employed as alternative cytotoxic proteins to the conventionally used ones such as ricin and Pseudomonas exotoxin . Mammalian RNases are attractive enzymes because of their comparable cytotoxicity when suitably directed and the likelihood of lower immunogenicity compared to plant and bacterial toxins . Bovine seminal RNase (BSRNase) is a member of the RNase superfamily, but differs in many interesting ways . Unlike the rest of the family it is dimeric, and possesses antitumor and immunosuppressive properties . These features make it a choice candidate for a single-chain antibody (scFv) based immunotoxin . This work describes preliminary data on the construction, expression in Escherichia coli and characterization of a tumor-specific scFv (directed against human placental alkaline phosphatase)-BSRNase chimeric molecule . It is shown that the created molecule has RNA degrading activity and antigen-binding activity when refolded from bacterial inclusion bodies.

Antonie Van Leeuwenhoek, 1994, 66(4), 307 - 12
Pyrimidine ribonucleoside catabolic enzyme activities of Pseudomonas pickettii; West TP; Pyrimidine ribonucleoside catabolic enzyme activities of the opportunistic pathogen Pseudomonas pickettii were examined . Of the pyrimidine and related compounds tested, only dihydrouracil (nitrogen source) and ribose (carbon source) supported growth . Thin-layer chromatographic separation of the uridine and cytidine catabolities produced by P . pickettii extracts indicated that this pseudomonad contained nucleoside hydrolase activity . Its presence was confirmed by enzyme assay . Hydrolase activity was elevated in both glucose- and ribose-grown cells relative to succinate-grown cells . Nucleoside hydrolase activity was depressed when dihydrouracil served as a nitrogen source . Cytosine deaminase activity was present in extracts prepared from succinate-, glucose- or ribose-grown cells when (NH4)2SO4 served as the nitrogen source although cells grown on glucose or ribose exhibited a higher enzyme activity . Cytosine deaminase activity was not detected in extracts prepared from cells grown on dihydrouracil as a nitrogen source . Both dihydropyrimidine dehydrogenase and dihydropyrimidinase activities were measurable in P . pickettii . The dehydrogenase activity was higher with NADH than with NADPH as its nicotinamide cofactor when uracil served as its substrate . Carbon source did not affect dehydrogenase or dihydropyrimidinase activity greatly but both activities were diminished in cells grown on the nitrogen source dihydrouracil.

Microbiol Immunol, 1994, 38(1), 1 - 9
Characterization of hemolytic and antifungal substance, cepalycin, from Pseudomonas cepacia; Abe M et al.; Hemolytic and antifungal substances, cepalycin I and cepalycin II, have been isolated from Pseudomonas cepacia JN106 . A large amount of cepalycins were produced by growing the cells on 1% glycerin-nutrient agar medium covered with a cellophane membrane . The cell-washed supernatant was applied to an Amberlite XAD2 column, and cepalycins were eluted with 70% ethanol containing 1mM HCl . Cepalycins were separated by reverse phase HPLC in two fractions which were designated as cepalycin I and cepalycin II . The two cepalycins have indistinguishable UV absorption spectra but have different levels of hemolytic activity relative to the UV absorption . From the inhibition of hemolytic activity of cepalycin by sterols, both cepalycins were suggested to interact with cholesterol in erythrocyte membrane . Such an interaction may contribute to their hemolytic and antifungal activities.

Bioconjug Chem, 1994 Jan-Feb, 5(1), 77 - 83
In vivo activities of acidic fibroblast growth factor-Pseudomonas exotoxin fusion proteins; Siegall CB et al.; Fibroblast growth factor receptors are highly expressed in a variety of cancer cells and activated vasculature . Using chimeric toxins targeted to cell-surface a FGF receptors, we have demonstrated specific cytotoxic activity to these cell types . These molecules, aFGF-PE40 and aFGF-PE40 KDEL, are fusion proteins containing acidic FGF and either a 40- or a 66-kDa binding defective form of Pseudomonas exotoxin, respectively . Both aFGF-toxin fusion proteins were able to inhibit protein synthesis in vitro in a variety of carcinoma cell lines . The half-life of aFGF-PE40 in serum was found to be 41 min when coadministered with heparin . Administration of aFGF-PE40 or aFGF-PE4E KDEL with heparin inhibits the growth of established KB and preestablished A431 epidermoid carcinoma xenografts in athymic mice . The antitumor activities of the two aFGF-toxin fusion proteins were equivalent against the KB tumor xenografts . While we were able to slow the growth of the KB tumor xenografts, we were unable to cause tumor regressions . Histochemical analysis of treated versus untreated tumor tissue revealed a difference in tumor size but not of vascularity . We conclude that aFGF-PE40 and aFGF-PE4E KDEL have in vivo antitumor activity that targets the tumor cell mass rather than vascular structures in mice xenografted with human epidermoid carcinoma.

Br J Cancer, 1994 Jan, 69(1), 32 - 9
Immunotoxins recognising a new epitope on the neural cell adhesion molecule have potent cytotoxic effects against small cell lung cancer; Zangemeister-Wittke U et al.; The present study describes a comparison of two potent immunotoxins which utilise an identical targeting component, a monoclonal antibody (SEN7) specific for small cell lung cancer (SCLC), conjugated to two different effector components, blocked ricin (bR) and Pseudomonas exotoxin A (PE) . SEN7 recognises a novel epitope on the neural cell adhesion molecule (NCAM) which is highly associated with SCLC . The immunotoxins SEN7-PE and SEN7-bR were selectively and potently active against a number of SCLC cell lines, of both classic and variant morphologies, inhibiting the incorporation of {3H}leucine with IC50 values ranging between 22 pM and 85 pM and between 7 pM and 62 pM for SEN7-PE and SEN7-bR respectively . Intoxication by both immunotoxins proceeded rapidly following short 2 h lag phases; the initial rates of protein synthesis inhibition occurred with t50 values of 6.5 h for SEN7-PE and 5.5 h for SEN7-bR . Monensin drastically enhanced the cytotoxic activity of the weakly active SEN7-ricin A-chain by 2,100-fold and of SEN7-bR by 80-fold but had no effect on SEN7-PE . In limiting dilution assays, four and more than 4.5 logs of clonogenic SW2 tumour cells were selectively eliminated from the cultures during continuous exposure to the immunotoxins SEN7-PE and SEN7-bR respectively, while antigen-negative cells required up to 1,000-fold more drug for a similar cell kill . SW2 cells surviving SEN7-bR treatment in the cultures did not express NCAM and consequently were not selectively killed by SEN7 immunotoxins . SW2 cells surviving continuous exposure to SEN7-PE showed no alteration in NCAM expression but were more resistant to intoxication mediated by PE . These cells were still sensitive to SEN7-bR.

Cancer Res, 1994 Jan 1, 54(1), 209 - 14
Comparison of two antibody-based methods for elimination of breast cancer cells from human bone marrow; Myklebust AT et al.; Three monoclonal antibodies reactive with antigens abundantly expressed on human carcinoma cells were used to develop and compare the efficacy of immunotoxins (ITs) and immunobeads for purging breast cancer cells from bone marrow . ITs constructed as conjugates of the monoclonal antibodies and Pseudomonas exotoxin A showed high specific cytotoxicity against three breast cancer cell lines, inhibiting protein synthesis by 50% at concentrations of 4 x 10(-13) M to 1 x 10(-10) M . Tested in a reproducible clonogenic assay, two of the ITs used at a concentration of 0.1 microgram/ml killed > 5 log units of MCF7 cells, the maximal sensitivity for assessing cytotoxic effects, and 1.5 log of T-47D tumor cells . At 1 microgram/ml, each of the three ITs eliminated > 5 log of both cell lines . The immunobead procedure removed 2.0-4.1 log of tumor cells with one purging cycle and up to 6.0 log with two cycles . The mixture of the three ITs or immunobeads was not clearly superior in efficacy, compared to the use of individual molecules, probably reflecting an overlap in expression of the respective antigens in these cell lines . For both methods, the purging efficacy was not reduced when the tumor cells were admixed with normal bone marrow cells at a ratio of 1:10 . The survival of colony-forming units, granulocyte/macrophage, was 49-86% with the immunobeads and 44-75% even at high concentrations (up to 2.5 micrograms/ml x 3) of the ITs . The results indicate that each of the two immunological methods can be safely used for effective elimination of tumor cells from the graft of breast cancer patients undergoing autologous bone marrow transplantation.

Carbohydr Res, 1993 Dec 28, 250(2), 275 - 87
Studies of O-specific polysaccharide chains of Pseudomonas solanacearum lipopolysaccharides consisting of structurally different repeating units; Kocharova NA et al.; The structures of the O-antigenic polysaccharide chains of lipopolysaccharides of a number of Pseudomonas solanacearum strains were elucidated mainly with the help of methylation analysis and 13C NMR spectroscopy, including a computer-assisted 13C NMR-based analysis . Six structurally distinct but related polysaccharides were identified . They have a backbone which is built up of three L-rhamnopyranosyl residues and one 2-acetamido-2-deoxy-D-glucopyranosyl residue, and is unsubstituted or substituted with a residue of L-xylopyranose or L-rhamnopyranose as a monosaccharide side chain . The lipopolysaccharides of most of the strains contain polysaccharide chains consisting of at least two structurally different types of repeating units . Three of the polysaccharides are common to more than one strain.

FEMS Microbiol Lett, 1993 Dec 15, 114(3), 339 - 42
Yeast genes involved in growth inhibition by Pseudomonas syringae pv . syringae syringomycin family lipodepsipeptides; Takemoto JY et al.; Saccharomyces cerevisiae genes encoding functions necessary for inhibition by the Pseudomonas syringae pv . syringae cyclic lipodepsipeptide, syringomycin-E, were identified by mutant analyses . Syringomycin-E-resistant mutants were isolated, shown to contain single recessive mutations, and divided into eight gene complementation groups . Representative strains from five groups were resistant to nystatin, and deficient in the plasma membrane lipid, ergosterol . All of the mutant strains were resistant to the related cyclic lipodepsipeptides, syringotoxin and syringostatin . The findings show that: 1) at least eight gene-encoded functions participate in the inhibitory response to syringomycin; 2) ergosterol is important for this response; 3) the three related lipodepsipeptides have similar modes of action.

Biochem J, 1993 Dec 15, 296 ( Pt 3), 867 - 75
Interaction of a 39 kDa protein with the low-density-lipoprotein-receptor-related protein (LRP) on rat hepatoma cells; Iadonato SP et al.; We have recently described a PAI-1-independent pathway of tissue-type plasminogen activator (t-PA) uptake and degradation on the rat MH1C1 hepatoma cell line . Further studies have implicated the low-density-lipoprotein-receptor-related protein (LRP) as the mediator of plasminogen-activator inhibitor type-1-independent t-PA endocytosis . The LRP is a multi-functional receptor which is shared by a variety of ligands, including alpha 2-macroglobulin, apoprotein E-enriched beta-very-low-density lipoprotein, t-PA and Pseudomonas exotoxin A . In each case, binding of ligand to this receptor can be inhibited by addition of the 39 kDa LRP-receptor-associated protein . This protein, which co-purifies with the LRP receptor, is the focus of our present study . 125I-labelled 39 kDa protein binds specifically and with high affinity to a single kinetic binding species on the rat MH1C1 cell surface . Scatchard analysis reveals an equilibrium dissociation constant (Kd) of 3.3 +/- 0.9 (S.D.) nM, with 380,000 +/- 190,000 (S.D.) binding sites per cell . Cross-linking studies indicate that the specific interaction between MH1C1 cells and the 39 kDa protein is mediated by an association with the LRP receptor . The 39 kDa protein strongly inhibits binding of 125I-t-PA, with an apparent Ki value of 0.5 nM . In addition, both unlabelled t-PA and 125I-labelled 39 kDa protein can be co-bound and cross-linked to the same cell-associated LRP receptor . Endocytosis of cell-surface-associated 39 kDa protein was shown to be rapid, with internalized ligand subsequently degraded and released to the extracellular milieu . The rate of uptake and degradation of 125I-labelled 39 kDa protein at 37 degrees C was determined to be 52 fmol/min per 10(6) cells, and supports a model for active recycling of the LRP receptor.

Zhonghua Yi Xue Za Zhi (Taipei), 1993 Dec, 52(6), 378 - 84
Pseudomonas septicemia in infants and children: a retrospective analysis of 49 cases; Wang TM et al.; Reviewed 49 cases of Pseudomonas bacteremia which occurred in pediatric patients during an 8-year period showed the annual rate, per 1000 discharges, was 1.7 . In most of the patients (73.5%), the disease was hospital-acquired . Male to female distribution was about 3 to 2 . The average age of the 49 patients was 1.29 years, and 55.1% were infected at less than 2 months old . Overall mortality was 44.9%; among the mortalities, 63.6% were under two months of age . Clinical features were not characteristic, but the most common sign associated with this infection was fever (42.9%) . Ecthyma gangrenosum occurred in only one patient . Respiratory tract and skin were the most frequent sources of the bacteremia . Polymicrobial bacteremia occurred in 18.4% . Patients with shock, pneumonia, inadequate antibiotic therapy or persistent neutropenia had a substantially poorer prognosis . Administration of combination therapy to patients with Pseudomonas bacteremia seemed to be superior to monotherapy for positive outcome.

FEMS Microbiol Lett, 1993 Dec 1, 114(2), 201 - 5
Purification of aspartate transcarbamoylase from Pseudomonas syringae; Shepherdson M et al.; The aspartate transcarbamoylase (ATCase) from Pseudomonas syringae has been purified . The purified enzyme was shown by SDS-PAGE to give two bands . Unambiguous results from N-terminal sequencing suggested that each band represented a homogeneous polypeptide . The M(r) (relative molecular mass) of the polypeptides was estimated to be 47 kDa and 34 kDa . The M(r) of the holoenzyme determined by gel filtration and electrophoretic migration in polyacrylamide gradient gels under non-denaturing conditions was estimated at approximately 490 kDa . These findings suggest a subunit structure different from any previously described for a bacterial ATCase.

Eur J Biochem, 1993 Dec 1, 218(2), 701 - 10
Cloning and characterization of the poly(hydroxyalkanoic acid)-depolymerase gene locus, phaZ1, of Pseudomonas lemoignei and its gene product; Jendrossek D et al.; Four different DNA fragments each coding for poly(hydroxyalkanoic acid) depolymerase (phaZ1-phaZ4) were isolated in pUC plasmids from a genomic library of Pseudomonas lemoignei in Escherichia coli . All recombinant strains secreted a highly active poly(3-hydroxybutyric acid) depolymerase and produced large translucent halos on an opaque medium containing poly(3-hydroxybutyric acid) granules . One DNA region (phaZ1) was present in seven independently isolated clones . Three other cloned DNA fragments were different from phaZ1 and from each other (phaZ2-phaZ4) . In phaZ1, an open-reading frame of 1245 bp was identified from the nucleotide sequence of a 5435-bp MboI fragment (57 mol G + C/100 mol) of this region and encoded a novel poly(hydroxyalkanoic acid) depolymerase of P . lemoignei, poly(3-hydroxybutyric acid) depolymerase C . A leader-sequence peptidase-cleavage site was predicted from the deduced amino acid sequence between Ala37 and Leu38 . The calculated relative molecular masses of the precursor and the putative mature protein were 43468 and 39581, respectively . The polypeptide contains a lipase consensus sequence (Gly-Xaa-Ser-Xaa-Gly) and an unusually high proportion of threonine residues (22 of 36 amino acids) near the C-terminus . The N-terminus of the deduced amino acid sequence of PhaZ1 differed from that of the purified poly(3-hydroxybutyric acid) depolymerases A, B and the poly(3-hydroxyvaleric acid) depolymerase of P . lemoignei . The phaZ1 gene product, poly(3-hydroxybutyric acid) depolymerase C, was partially purified from recombinant E . coli (pUC91::phaZ1) . The purified protein was specific for poly(hydroxyalkanoic acid) consisting of monomers of four or five carbon atoms and for p-nithrophenylbutyrate as substrates . The polymer-hydrolyzing activity, but not the p-nitrophenylate esterase activity, was inhibited by complex media such as Luria-Bertani medium and by soluble E . coli proteins . The enzyme protein did not cross-react with antibodies raised against purified poly(3-hydroxyvaleric acid) depolymerase of P . lemoignei.

Eur J Immunol, 1993 Dec, 23(12), 3181 - 8
Selection of internalization-deficient cells by interleukin-2-Pseudomonas exotoxin chimeric protein: the cytoplasmic domain of the interleukin-2 receptor beta chain does not contribute to internalization of interleukin-2; Furse RK et al.; To study the structural basis of ligand-induced receptor-mediated internalization of interleukin-2 (IL-2), a strategy has been developed to generate variant T cells that are deficient in internalization of this cytokine . IL-2 receptor (IL-2R) alpha- and beta-bearing EL4 cells, that express high-affinity IL-2R and internalize IL-2, were treated with low doses of IL-2-Pseudomonas exotoxin chimeric protein (IL-2-PE40) . This treatment resulted in isolation of a variant (CX1) that was unable to express high-affinity IL-2R or internalize IL-2 . Transfection of CX1 with the IL-2R beta cDNA led to surface expression of IL-2R beta and high-affinity IL-2R as well as the ability to internalize IL-2 . This finding indicates that the absence of the beta subunit was the sole defect in CX1 responsible for its failure to internalize IL-2 . By transfecting CX1 with mutated beta cDNA, several CX1 transfectants were produced that expressed a beta-subunit that lacked all amino acids of the intracytoplasmic region . These transfectants expressed high-affinity IL-2R and internalized IL-2 at a rate comparable to cells expressing wild-type beta-chain . These results demonstrate that internalization of IL-2 is independent of any signals contained in the intracytoplasmic tail of the beta subunit and raise the possibility that such signals may be entirely contained within the gamma subunit.

J Urol, 1993 Dec, 150(6), 1984 - 9
Long-term effects of ureteric stent after ureteric dilation; Selmy GI et al.; Balloon dilation of the right ureterovesical junction (UVJ) and distal ureter to three times its normal caliber was performed in 12 pigs . A right double-J (D-J) stent was inserted after dilation in 6 pigs . Bilateral upper tract dynamics with different perfusion rates (0.5, 2 and 4 ml . per minute) were recorded before dilation, immediately after dilation, and then 4 and 7 weeks after dilation . Immediate and late antegrade nephrostograms as well as suprapubic cystograms were taken . Grade 3 reflux occurred in 100% of animals at 7 weeks on the dilated, stented ureter and no reflux on the dilated, nonstented ureter . At 7 weeks on the dilated, stented side, significant growth (> 100,000, colonies) of Pseudomonas species was noted in all animals . Creatinine clearance was significantly reduced on the dilated, stented side when compared to the dilated, nonstented side at 7 weeks . Histologic examination of the dilated, stented and dilated, nonstented ureters at 4 weeks revealed a segmental muscular defect with muscular regeneration starting from the edge of the defect, particularly in the innermost region . At 7 weeks, there was a more advanced, but similar, pattern of muscular regeneration in both groups . However, at 7 weeks, metaplastic changes of the ureter and chronic pyelonephritis were evident on the dilated, stented ureter . Electron microscopy showed that myofibroblasts played a major role in the healing process with new muscle formation . At 4 weeks, no significant morphologic difference was found between the dilated, stented and dilated, nonstented ureters . At 7 weeks, however, it appeared that the ureteric stent resulted in damage and deterioration of renal function without affecting muscular regeneration of the ureter . We conclude that the changes observed could be entirely due to the infection associated with the stent rather the stent itself.

J Urol, 1993 Dec, 150(6), 1950 - 5
In vitro sensitivity testing of human bladder cancers and cell lines to TP-40, a hybrid protein with selective targeting and cytotoxicity; Sarosdy MF et al.; TP-40 is a hybrid fusion protein produced by recombinant technology and consists of a molecule of transforming growth factor-alpha (TGF-alpha) fused to the Pseudomonas exotoxin PE-40 . A panel of human and murine bladder cancer cell lines was found to be universally sensitive in vitro to TP-40 in a clonogenic assay . All lines expressed receptor for epidermal growth factor (EGF), though none demonstrated gene amplification for the EGF receptor . The sensitivity to TP-40 may be blocked by preexposure to EGF . Six human bladder tumors taken directly from patients were all sensitive in vitro to TP-40; these included well-differentiated tumors . TP-40 may prove to be effective as an intravesical agent in bladder cancer via selective targeting to cells that express EGF receptors, as do the majority of human bladder cancers.

J Biochem (Tokyo), 1993 Dec, 114(6), 930 - 5
An inducible NADP(+)-dependent D-phenylserine dehydrogenase from Pseudomonas syringae NK-15: purification and biochemical characterization; Packdibamrung K et al.; An inducible NADP(+)-dependent D-phenylserine dehydrogenase {EC 1.1.1.-}, which catalyzes the oxidation of the hydroxyl group of D-threo-beta-phenylserine, was purified to homogeneity from a crude extract of Pseudomonas syringae NK-15 isolated from soil . The enzyme consisted of two subunits identical in molecular weight (about 31,000) . In addition to D-threo-beta-phenylserine, it utilized D-threo-beta-thienylserine, D-threo-beta-hydroxynorvaline, and D-threonine as substrates but was inert towards other isomers of beta-phenylserine and threonine . It showed maximal activity at pH 10.4 for the oxidation of D-threo-beta-phenylserine, and it required NADP+ as a natural coenzyme . NAD+ showed a slight coenzyme activity . The enzyme was inhibited by p-chloromercuribenzoate, HgCl2, and monoiodoacetate but not by the organic acids such as tartronate . The Michaelis constants for D-threo-beta-phenylserine and NADP+ were 0.44 mM and 29 microM, respectively . The N-terminal 27 amino acids sequence was determined . It suggested that the NADP(+)-binding site was located in the N-terminal region of the enzyme.

Indian J Biochem Biophys, 1993 Dec, 30(6), 405 - 10
Potentiation of ricin cytotoxicity by liposomal monensin under in vitro and in vivo conditions; Madan S et al.; Effect of monensin, intercalated in liposomes on the cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in phagocytic and non-phagocytic cells as well as in mice has been studied . Intercalation does not disturb the integrity of the liposomal bilayer and substantially enhances the cytotoxicities of ricin and Pseudomonas exotoxin A in both phagocytic and non-phagocytic cells while it has no effect on diphtheria toxin . The observed effect is highly dependent on the liposomal lipid composition as well as cell types . The potentiating ability of monensin in neutral vesicle is 2.2-fold higher than in negatively charged vesicles in non-phagocytic cells while no difference was observed in phagocytic cells . Incorporation of stearylamine in liposomes reduces the potentiating effect of monensin . Liposomal monensin has also been found to enhance the cytotoxicity of ricin in mouse in vivo in a dose-dependent manner and is maximal when ricin is injected within 60 min of monensin injection . Liposomal monensin remains in circulation for 2 hr while free monensin remains only for 15 min . Tissue distribution studies reveal that liposomal monensin is present mainly in the liver and spleen which are also the major sites for ricin accumulation . Thus liposome is found to be an effective delivery vehicle for monensin to potentiate the cytotoxicity of immunotoxins or hormonotoxins and could prove useful for selective elimination of cancer cells.

Appl Environ Microbiol, 1993 Dec, 59(12), 4180 - 8
DNA sequence variation and phylogenetic relationships among strains of Pseudomonas syringae pv . syringae inferred from restriction site maps and restriction fragment length polymorphism; Legard DE et al.; We evaluated the restriction fragment length polymorphism of genomic DNA among 53 strains of the phytopathogenic bacterium Pseudomonas syringae pv . syringae . Twenty-nine strains were isolated from beans, and the rest were isolated from 11 other hosts . Southern blots of DNA digested with EcoRI or HindIII were hybridized to two random probes from a cosmid library of P . syringae pv . syringae and a hrp (hypersensitive reaction and pathogenicity) cluster cloned from P . syringae pv . syringae . The size of hybridizing fragments was determined, and a similarity matrix was constructed by comparing strains on a pairwise basis for the presence or absence of fragments . The proportion of shared fragments was then used to estimate sequence divergence . Dendrograms were produced by using the unweighted pair group method with averages and the neighbor-joining method . For the hrp region, BamHI, EcoRI, EcoRV, and HindIII restriction sites were mapped for six representative bean strains and used to construct EcoRI and HindIII restriction maps for all 30 strains pathogenic on beans . Restriction mapping revealed the presence of a 3-kb insertion in nine bean strains and a probable second insertion or deletion event on the left-hand side of the hrp cluster that biased estimates of nucleotide sequence divergence from fragment comparisons . This demonstrated that the determination of phylogenetic relationships among bacteria by using restriction fragment length polymorphism data requires mapping restriction sites to remove the effect of insertion or deletion events on the analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Asian Pac J Allergy Immunol, 1993 Dec, 11(2), 149 - 54
Application of indirect immunofluorescence microscopy to colony identification of Pseudomonas pseudomallei; Naigowit P et al.; Indirect immunofluorescence microscopy was used as a colony identification method of Pseudomonas pseudomallei isolates . The antisera against lipopolysaccharide and protein fractions of P . pseudomallei were prepared in guinea pigs and rabbits . With these antisera and fluorescence-labelled anti-guinea pig IgG and anti-rabbit IgG prepared in sheep (goat), indirect immunofluorescence microscopy was conducted on the colonies of P . pseudomallei and other species of bacteria . The overall results indicated that this method is efficient, rapid and specific for identification of P . pseudomallei colonies from clinical specimens.

Kansenshogaku Zasshi, 1993 Dec, 67(12), 1160 - 6
{Epidemiological study on nosocomial infection of Pseudomonas cepacia by plasmid analyses}; Yamagishi Y et al.; In order to clarify the usefulness of plasmid analyses in epidemiological study, plasmid DNA profiles, restriction fragment patterns of chromosomal DNA analysed by pulsed-field gel electrophoresis (PFGE), and patterns of extracellular products were performed in twenty-four nosocomial strains of Pseudomonas cepacia isolated at the Kagawa Medical School Hospital . Fifteen strains were obtained from 15 patients admitted in A ward, three strains were obtained from nebulizer devices used in A ward, 5 strains were obtained from 5 patients admitted in B ward, and 1 strain was obtained from a patient admitted in C ward . Three different patterns which differed from ward to ward were clearly distinguished by PFGE patterns and patterns of extracellular products . On the other hand, plasmid DNA profiles were different in some strains obtained from B ward . These results suggested that plasmid analyses combined with other typing methods are useful in more precise epidemiological analysis on nosocomial infection of P . cepacia.

Science, 1993 Nov 26, 262(5138), 1432 - 6
Map-based cloning of a protein kinase gene conferring disease resistance in tomato; Martin GB et al.; The Pto gene in tomato confers resistance to races of Pseudomonas syringae pv . tomato that carry the avirulence gene avrPto . A yeast artificial chromosome clone that spans the Pto region was identified and used to probe a leaf complementary DNA (cDNA) library . A cDNA clone was isolated that represents a gene family, at least six members of which genetically cosegregate with Pto . When susceptible tomato plants were transformed with a cDNA from this family, they were resistant to the pathogen . Analysis of the amino acid sequence revealed similarity to serine-threonine protein kinases, suggesting a role for Pto in a signal transduction pathway.

J Biol Chem, 1993 Nov 25, 268(33), 24847 - 54
Cytotoxicity of folate-Pseudomonas exotoxin conjugates toward tumor cells . Contribution of translocation domain; Leamon CP et al.; Folate-protein conjugates can be nondestructively delivered into a cell's cytoplasm via folate receptor-mediated endocytosis if (i) the target cells express a folate-binding protein, and (ii) if the folate is linked to its attached protein at a site that does not interfere with receptor recognition . Because such conjugates have been observed to remain in endosomal compartments for extended periods following cellular uptake, we decided to evaluate whether release into the cytoplasm might be expedited by inclusion of a translocation domain in the folate-protein construct . To test this possibility, momordin-folate and truncated Pseudomonas exotoxin-folate conjugates (LysPE38 and Cys-PE35), i.e . protein synthesis inhibitors either lacking or containing the desired translocation domain, respectively, were examined for their abilities to block protein synthesis in a variety of cell types . The translocation competent LysPE38-folate construct was found to kill cells six times more rapidly with 10-fold greater potency than the permeation-incompetent mormordin-folate . Further, cells expressing low levels of folate receptors could only be exterminated by the translocation competent Pseudomonas exotoxin-folate conjugates . When the translocation capability of CysPE35-folate was inactivated by modification of Cys287, the construct also lost most of its cytotoxicity . These data suggest that autocatalysis of transport from an internal vesicular compartment into the cytoplasm can greatly augment the cytotoxicity of a protein toxin entering cells via the folate endocytosis pathway . Because the folate ligand can selectively target a protein conjugate to cancer cells in the presence of normal cells, such translocatable toxin-folate constructs warrant further study as a possible treatment for some malignancies.

Biochemistry, 1993 Nov 16, 32(45), 12245 - 50
Specificity of 4-chlorobenzoyl coenzyme A dehalogenase catalyzed dehalogenation of halogenated aromatics; Liang PH et al.; Steady-state and transient kinetic techniques were used to evaluate the efficiency of 4-chlorobenzoyl coenzyme A (4-CBA-CoA) turnover catalyzed by 4-CBA-CoA dehalogenase from Pseudomonas sp . CBS-3 . The kcat for a single turnover on the enzyme was found to be 2 s-1, while that for multiple turnovers was found to be 0.6 s-1 . Catalysis rather than product release was judged to be rate limiting . Comparison of the rates of turnover of 4-bromobenzoyl-CoA (1.4 s-1), 4-iodobenzoyl-CoA (1.1 s-1), and 4-fluorobenzoyl-CoA (8 x 10(-6) s-1) indicated that cleavage of the carbon-halogen bond occurs in the rate-limiting transition state of the reaction . Structure-activity measurements made with 4-CBA-CoA analogs bearing electron-donating or -withdrawing substituents at C(2) or C(3) suggested the importance of steric/solvation effects on the enzymatic reaction and failed to provide insight into the nature of the reaction intermediate . The inhibition constants measured for benzoyl-CoA (72 microM), CoA (140 microM), and 4-chlorobenzoate (21 mM) compared to the Km measured for 4-CBA-CoA (4 microM) suggest the dominant role played by the CoA moiety in substrate anchoring.

Bioconjug Chem, 1993 Nov-Dec, 4(6), 581 - 5
Purification and characterization of IL6-PE4E, a recombinant fusion of interleukin 6 with Pseudomonas exotoxin; Kreitman RJ et al.; We have developed a procedure to purify the recombinant fusion toxin IL6-PE4E from Escherichia coli which results in a high yield of fully active monomeric protein of high purity and very low endotoxin content . The chimeric toxin is composed of human interleukin 6 (IL6) fused to a derivative of Pseudomonas exotoxin (PE) containing mutations in the binding domain which prevent binding to the PE receptor . In a typical preparation, 20 g of E . coli cells expressing the plasmid encoding IL6-PE4E were treated with lysozyme and washed repeatedly with detergent (Triton X-100), to obtain 500 mg of inclusion bodies . The recombinant protein was denatured and reduced in guanidine hydrochloride solution containing dithioerythritol and refolded in a redox buffer containing oxidized glutathione and L-arginine . After purification of the dialyzed protein by anion-exchange, polymyxin B, and sizing chromatography, we obtained 100 mg (20% of recombinant protein) of purified monomer with 0.6-2.5 endotoxin units/mg of protein . Amino terminal sequencing confirmed the first 20 amino acids . IL6-PE4E purified in this manner was fully cytotoxic toward human multiple myeloma, hepatoma, epidermoid carcinoma, and prostate carcinoma cell lines . After intravenous injection into mice, we found the dose-limiting toxicity to be to the liver, by measurement of serum transaminases and histologic evaluation of the liver . The LD50 was 450 micrograms/kg . We conclude that IL6-PE4E can be purified efficiently for preclinical testing.

Clin Infect Dis, 1993 Nov, 17(5), 877 - 80
Infections caused by Pseudomonas pickettii in association with permanent indwelling intravenous devices: four cases and a review; Raveh D et al.; Permanent indwelling intravenous devices (PIIDs) have become increasingly prevalent in the past decade . These devices offer the advantage of long-term and convenient venous access, but their not-infrequent colonization by bacterial or fungi can lead to bloodstream infection with or without sepsis . We describe a series of four patients with PIIDs who became infected with Pseudomonas pickettii, and we review the properties of this unusual organism and the clinical presentation of the infections it causes.

Exp Lung Res, 1993 Nov-Dec, 19(6), 671 - 84
Most of the lipid in purulent sputum is bound to mucus glycoprotein; Nadziejko CE et al.; Mucus glycoprotein (mucin) is the principal biochemical constituent of sputum . Appreciable quantities of lipid, DNA, and nonmucin proteins are also present, particularly in purulent sputum . Previous studies have shown that purified mucin from respiratory tract secretions contains non-covalently bound lipid . However, it is not known whether lipids in purulent sputum are associated only with mucin or with nonmucin proteins and DNA as well . Purulent sputum was obtained from cystic fibrosis patients . Tracheal aspirates were obtained from noncystic patients with purulent secretions due to Pseudomonas species, as well as from noninfected, noncystic patients who had mucoid airway secretions . The lipid content of unfractionated airway secretions (sputum or tracheal aspirates), gel filtration-purified mucin, and nonmucin components of the airway secretions was analyzed . The purified mucin from all three groups had a significantly higher content of solvent-extractable lipid as compared to unfractionated airway secretions . The nonmucin fractions contained only small amounts of lipid . Density gradient centrifugation verified that the lipid recovered in the purified mucin fraction was complexed with the glycoprotein . The results of this study indicate that most of the lipids in purulent sputum are associated with mucin.

Plant J, 1993 Nov, 4(5), 813 - 20
Identification of a disease resistance locus in Arabidopsis that is functionally homologous to the RPG1 locus of soybean; Innes RW et al.; A new disease resistance locus in Arabidopsis, RPS3, was identified using a previously cloned avirulence gene from a non-Arabidopsis pathogen . The avrB avirulence gene from the soybean pathogen Pseudomonas syringae pv . glycinea was transferred into a P . syringae pv . tomato strain that is virulent on Arabidopsis, and conversion to avirulence was assayed on Arabidopsis plants . The avrB gene had avirulence activity on most, but not all, Arabidopsis ecotypes . Of 53 ecotypes examined, 45 were resistant to a P . syringae pv . tomato strain carrying avrB, and eight were susceptible . The inheritance of this resistance was examined using crosses between the resistant ecotype Col-0 and the susceptible ecotype Bla-2 . In F2 plants from this cross, the ratio of resistant:susceptible plants was approximately 3:1, indicating that resistance to P . syringae expressing avrB is determined by a single dominant locus in ecotype Col-0, which we have designated RPS3 . Using RFLP analysis, RPS3 was mapped to chromosome 3, adjacent to markers M583 and G4523, and < or = 1 cM from another disease resistance locus, RPM1 . In soybean, resistance to P . syringae strains that carry avrB is controlled by the locus RPG1 . Thus, RPG1 and RPS3 both confer avrB-specific disease resistance, suggesting that these genes may be homologs.

Am J Emerg Med, 1993 Nov, 11(6), 606 - 8
Contact lens abrasions and the nonophthalmologist; Schein OD; Corneal abrasions associated with contact lens wear are commonly evaluated and treated in acute care clinics and emergency departments by nonophthalmologists . The risk of progression to suppurative keratitis in this setting requires management distinct from that of other mechanical (eg, fingernail scratch) corneal abrasions . The antibiotic chosen should reflect the need for prophylaxis against Pseudomonas . Conditions favoring bacterial growth, specifically occlusive patching and/or use of steroid containing compounds, should be avoided, and 24-hour follow-up examination is recommended.

Am Rev Respir Dis, 1993 Nov, 148(5), 1266 - 71
Passive smoking and lung function in cystic fibrosis; Kovesi T et al.; The relationship between passive exposure to cigarette smoking and objective measures of health was examined in 340 patients with cystic fibrosis attending a large hospital-based clinic . Patients who came from households with smokers did not differ from those living in smoke-free households in terms of nutritional status, clinical scores, spirometry, or colonization with Pseudomonas . The number of cigarettes smoked in the household was not significantly related to nutritional status, clinical score, spirometry, or hospitalization . Similar results were found when children 6 to 11 yr of age were analyzed separately, except that height percentile was negatively related to the number of cigarettes smoked in the household . The effects of household exposure to cigarette smoke were further evaluated by analyzing changes in nutritional status, clinical score, and spirometry over a 15-yr period among patients whose families never, always, stopped, or started smoking during this time . Height percentile increased slightly during this interval among those whose households never smoked, whereas no change occurred among patients whose households always smoked, and a decline was seen among patients whose households quit . These differences were statistically significant . Patients whose households never smoked had consistently higher pulmonary function measurements than did patients whose families always smoked, although the differences were not statistically significant . The rates of decline were similar in these two groups . Patients whose households stopped smoking had significantly lower pulmonary functions at the end of the study than did subjects whose households never smoked.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 9906 - 10
Leucine aminopeptidase: an inducible component of the defense response in Lycopersicon esculentum (tomato); Pautot V et al.; A leucine aminopeptidase (EC 3.4.11.1) cDNA clone (DR57) that was induced in response to Pseudomonas syringae pv . tomato (P.s . tomato) infection was isolated using a subtractive hybridization-enriched cDNA probe . Genomic DNA blot analysis showed that the tomato genome had two leucine aminopeptidase genes . The levels of DR57 mRNAs after P.s . tomato infection and mechanical wounding were determined in two inbred tomato lines that exhibit susceptibility and resistance to P.s . tomato . DR57 mRNAs were detected 12 hours after infection and 4 hours after wounding . Furthermore, DR57 mRNAs were systemically induced in response to wounding . DR57 mRNAs were induced in leaves after Spodoptera littoralis feeding but were not detected in detached leaf controls . Possible roles for the DR57 leucine aminopeptidase in the defense reactions are discussed.

J Bacteriol, 1993 Nov, 175(22), 7216 - 21
Kinetics of appearance and disappearance of classes of bacterial ice nuclei support an aggregation model for ice nucleus assembly; Ruggles JA et al.; The kinetics of appearance and disappearance of three classes of ice nuclei in Pseudomonas syringae was investigated under conditions where high-level expression of the ice nucleation phenotype was obtained . The appearance of types 1, 2, and 3 ice nuclei, catalyzing nucleation at -2 to -5, -5 to -7, and -7 to -10 degrees C, respectively, was investigated during low-temperature induction in wild-type strains and in a unique, detergent-sensitive mutant that contained no type 3 ice nuclei when grown at 32 degrees C . Nuclei appeared in the following order: type 3, then type 2 and type 1 . The disappearance of classes of ice nuclei was monitored during high-temperature treatment of fully induced cells; nuclei disappeared in the order type 1, type 2, and type 3 . Although analysis of nucleation events is complicated by masking and unmasking of ice sites in the same cells, these temporal sequences of ice nucleus appearance or disappearance are consistent with an aggregation model for ice nucleus assembly (A . G . Govindarajan and S . E . Lindow, Proc . Natl . Acad . Sci . USA 85:1334-1338, 1988; G . Warren and P . Wolber, Mol . Microbiol . 5:239-243, 1991).

Eur J Biochem, 1993 Nov 1, 217(3), 1011 - 7
Thermodynamics of the maleate and citraconate hydration reactions catalysed by malease from Pseudomonas pseudoalcaligenes; van der Werf MJ et al.; Malease from Pseudomonas pseudoalcaligenes catalyses the hydration of both maleate and citraconate to D-malate and D-citramalate, respectively . The Kapp for these hydration reactions were 2050 and 104, respectively, under standard biochemical conditions (25 degrees C, pH 7.0, I = 0.1) . The influence of the pH (6.0-8.5) on Kapp was determined . The Gibbs-free-energy changes under standard biochemical conditions for the hydration of the dianionic acids were calculated to be -19.28 kJ.mol-1 and -11.65 kJ.mol-1, respectively . From the obtained data together with data from the literature, the Gibbs free energy of formation of maleate2- and citraconate2- were calculated to be -588.91 kJ.mol-1 and -600.56 kJ.mol-1, respectively . The influence of the temperature (10-40 degrees C) on Kapp was determined for both hydration reactions . The enthalpy change (delta H degrees') and entropy change (delta S degrees') under standard biochemical conditions for the maleate2- (delta H degrees' = 18.07 kJ.mol-1, delta S degrees' = 2.94 J.mol-1 x K-1) and citraconate2- (delta H degrees' = -22.55 kJ.mol-1, delta S degrees' = -35.92 kJ.mol-1 x K-1) hydration reactions were calculated . The reaction rate of malease from Ps . pseudoalcaligenes was studied for both hydration reactions as a function of temperature . From these studies, the Gibbs free energies of activation for the maleate and citraconate hydration reactions catalysed by malease from Ps . pseudoalcaligenes were calculated to be 62.21 kJ.mol-1 and 63.43 kJ.mol-1, respectively.

Zhonghua Zhong Liu Za Zhi, 1993 Nov, 15(6), 419 - 22
{Specific cytotoxicity of recombinant interleukin 6-pseudomonas exotoxin fusion protein (IL6-PE40) on LT12 leukemic cells expressing high levels of interleukin 6 receptor (IL6R)}; Xi YZ; An acute promyelocytic leukemic cell line LT12 was transduced with IL6R cDNA by electroporation . The resultant LT12-IL6R+ leukemic cells expressed more than 1000 IL6R per cell . The effect of the recombinant IL6-PE40 on both the LT12-IL6R+ cells and the IL6R- parental cells (LT12-IL6R-) was studied by leukemic progenitor colony (CFU-L) formation and 3H-TdR incorporation assays . It was found that IL6-PE40 at concentrations ranging from 1 to 1,000 ng/ml inhibited CFU-L formation and DNA synthesis of LT12-IL6R+ cells in a dose-dependent manner . No inhibition was seen at the same concentration range in LT12-IL6R- cells . At concentrations of 250-1000ng/ml, IL6-PE40 led to only 40% inhibition of DNA synthesis in LT12-IL6R- cells . Inasmuch as rIL6 at the same concentrations had no significant effect on both IL6R+ and IL6R- cells, we concluded it is IL6-PE40 that exerts its highly specific killing on leukemic cells expressing high levels of IL6R.

Anal Biochem, 1993 Nov 1, 214(2), 500 - 5
Ortho-anisic acid as internal standard for the simultaneous quantitation of salicylic acid and its putative biosynthetic precursors in cucumber leaves; Meuwly P et al.; Salicylic acid (SA) acts as an endogenous signal for the induction of systemic acquired resistance (SAR) in plants infected by pathogens . In order to study SA biosynthesis in plants, a quantitative method using an internal standard was developed for the simultaneous measurement of SA and three of its putative bioprecursors, namely trans-cinnamic acid (CA), trans-ortho-hydroxycinnamic acid (oHCA), and benzoic acid (BA) . ortho-Anisic acid (oANI) was found to be a suitable internal standard for all four compounds . It was not present as endogenous compound in cucumber leaves, no substance interfered with its detection, and it followed identical losses as SA, CA, oHCA, and BA during the extraction procedure . Baseline separation of these four compounds and of eight other plant phenolics was achieved in 35 min using deactivated reversed-phase HPLC . On-line detection was performed fluorimetrically for SA, oANI, and oHCA (with the excitation and emission wavelengths optimized for each compound) and with uv detection for CA and BA . Correlation equations for SA, CA, oHCA, and BA using oANI as internal standard were linear for a broad range of concentrations . The limits of detection (in ng/g fresh weight, FW) obtained during routine analyses were as follows: SA, 4; oHCA, 100; CA, 150; and BA, 500 . The method was used to quantify levels of free and acid-hydrolyzed bound SA in cucumber plants either 5 days postinoculation with Pseudomonas lachrymans or after mock-inoculation with water on their first leaf.(ABSTRACT TRUNCATED AT 250 WORDS)

J Pediatr, 1993 Nov, 123(5), 745 - 7
Source of Pseudomonas cepacia: ribotyping of isolates from patients and from the environment; Fisher MC et al.; Ribotyping, an analysis of bacterial strain identity based on chromosomal restriction fragment length polymorphisms, was performed on 68 isolates of Pseudomonas cepacia recovered from patients receiving care at the two children's hospitals in Philadelphia and from environmental samples . Twenty different ribotypes were identified . Ribotype R3 predominated among isolates from all three sources . These findings are consistent with the acquisition of P . cepacia from the environment or by person-to-person transmission.

Bioorg Med Chem, 1993 Nov, 1(5), 375 - 80
Enzymic acylation of methyl D- and L-glycopyranosides: influence of the 3-hydroxyl group; Colombo D et al.; Porcine pancreatic (PPL), Candida cylindracea (CCL) and Pseudomonas cepacia (LPS) lipases, suspended in organic solvents, were used to regioselectively acylate methyl 6-O-butyryl-alpha-D- and L-allopyranosides and methyl 6-O-butyryl-3-deoxy-alpha-D- and L-ribo-hexopyranosides . Both the D- and the L-3-deoxy sugars showed a complete regioselectivity, while the reactions of the allosides proved to be less regioselective . This indicates that the presence of the hydroxyl group at C-3 is an unfavourable factor for the action of the lipases.

J Infect, 1993 Nov, 27(3), 301 - 4
Chronic granulomatous disease presenting in childhood with Pseudomonas cepacia septicaemia; Lacy DE et al.; Two children who presented with fever, enlarged liver and spleen and ascites were found to have Pseudomonas cepacia septicaemia which proved fatal despite appropriate antibiotics and maximum supportive care . Chronic granulomatous disease of childhood (CGD) was subsequently diagnosed in both children . The possibility of CGD needs to be considered in any child with unexplained P . cepacia infection.

Bioconjug Chem, 1993 Nov-Dec, 4(6), 483 - 9
Basic fibroblast growth factor-Pseudomonas exotoxin chimeric proteins; comparison with acidic fibroblast growth factor-Pseudomonas exotoxin; Gawlak SL et al.; We have constructed growth factor-toxin chimeric molecules composed of basic fibroblast growth factor (bFGF) and two different binding mutant forms of Pseudomonas exotoxin termed bFGF-PE40 and bFGF-PE4E KDEL . The chimeric molecules were expressed in Escherichia coli and localized to both inclusion bodies and the spheroplast cytoplasm . The bFGF-toxin fusion protein that was isolated and purified from inclusion bodies was 3-fold more active in inhibiting protein synthesis than that purified from spheroplast cytoplasm . Immunoreactivity of purified bFGF-toxin fusion protein to anti-bFGF antibodies was similar to that of native bFGF, as determined by ELISA analysis . A variety of carcinoma cell lines were sensitive to bFGF-PE40 and bFGF-PE4E KDEL, including H3396 (breast), Hep G2 (hepatocellular), and A431 (epidermoid) . The concentration of chimeric toxin that inhibited protein synthesis by 50% (EC50) was 110, 70, and 18 ng/mL for bFGF-PE40 and 15, 1, and 18 ng/mL for bFGF-PE4E KDEL . In comparison with fusion-toxins composed of acidic fibroblast growth factor (aFGF) and either PE40 or PE4EKDEL, bFGF-PE40 and bFGF-PE4E KDEL were similarly cytotoxic on most cell lines tested . Human aortic smooth muscle cells were sensitive to both bFGF and aFGF toxin fusion proteins . However, human aortic endothelial cells were sensitive to the bFGF-toxins but were resistant to both aFGF-toxin forms . Time course studies showed that bFGF-PE40 needed a 4-6-h exposure to target cells for peak inhibition of protein synthesis on both MCF-7 and A431 cells, while aFGF-PE40 was almost fully active within a 2-h incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioorg Khim, 1993 Nov, 19(11), 1089 - 94
{Antigenic polysaccharides of bacteria 38 . Structure of O-antigens of Pseudomonas solanacearum ICMP 7942 and ICMP 8169}; Shashkov AS et al.; The structures I and II were established for the O-antigens of Pseudomonas solanacearum ICMP 7942 and ICMP 8169, respectively, by means of NMR-spectroscopy, including 2D homonuclear shift-correlated spectroscopy (COSY), rotating-frame NOE spectroscopy (ROESY) and heteronuclear 13C, 1H multiquantum correlation spectroscopy (HMQC) . In the polysaccharide of the ICMP 8169 strain substitution of the main chain by the xylose residue is non-stoichiometric, i.e . this antigen contains also the repeating units of the polysaccharide of the ICMP 7942 strain (the ratio II: I is approximately 3:1) . {formula: see text}

Kansenshogaku Zasshi, 1993 Nov, 67(11), 1115 - 25
Nosocomial outbreak of Pseudomonas cepacia respiratory infection in immunocompromised patients associated with contaminated nebulizer devices; Takigawa K et al.; From May 1990 to August 1991, 36 patients admitted to the Department of Internal Medicine in a medical school hospital with hematological malignancies or solid tumors, developed respiratory tract colonization with Pseudomonas cepacia . Sixteen (44.4%) of these patients developed pneumonia, and four (11.1%) died of respiratory failure due to P . cepacia pneumonia . Extensive survey of the hospital environment as well as equipment showed that nebulizer devices used by the patients for inhalation were contaminated with P . cepacia . Phenotypic characteristics, (production of hemolysin and extracellular enzymes {lipase, lecithinase and protease}), the Analytical Profile Index 20 NE pattern, and the pattern of DNA fingerprinting by pulse-field gel electrophoresis in clinically isolated strains and strains derived from nebulizer devices were compared . The strains of P . cepacia obtained from patients in the Department of Internal Medicine were indistinguishable from each other and also from those isolated from nebulizer devices, but were different from those isolated from patients in other departments at the same time . These results demonstrated that the outbreak of P . cepacia respiratory colonization in immunocompromised patients was a nosocomial acquisition, and probably occurred by transmission through contaminated nebulizer devices.

J Clin Microbiol, 1993 Nov, 31(11), 3017 - 22
Epidemic of Pseudomonas cepacia in an adult cystic fibrosis unit: evidence of person-to-person transmission; Smith DL et al.; An epidemic of Pseudomonas cepacia occurred in an adult cystic fibrosis center in the United Kingdom, despite a policy of segregation of infected and noninfected patients within the hospital . Investigation of the outbreak by ribotyping and pulsed-field gel electrophoresis to characterize P . cepacia strain genomes together with inquiry into social contacts between patients revealed evidence of person-to-person transmission outside the hospital environment . Segregation policies aimed at reducing the spread of this infection in the cystic fibrosis community need to encompass patient contacts outside the hospital environment.

Gene, 1993 Oct 29, 133(1), 31 - 8
Characterization of the genes controlling the biosynthesis of the polyketide phytotoxin coronatine including conjugation between coronafacic and coronamic acid; Bender CL et al.; Pseudomonas syringae pv . glycinea PG4180 produces a chlorosis-inducing phytotoxin, coronatine (COR), which consists of a polyketide component, coronafacic acid (CFA), which is coupled via amide bond formation to coronamic acid (CMA), an ethylcyelopropyl amino acid (aa) derived from isoleucine . P . syringae pv . syringae strains PS51 and PS61, which do not synthesize coronafacoyl compounds (conjugates between CFA and aa), acquired the ability to produce CFA and COR when transformed with p4180A, a 90-kb indigenous plasmid in PG4180 . Tn5 mutagenesis indicated that the COR biosynthetic genes in PG4180 are clustered within a 30-kb region on p4180A . The phenotype of selected COR-defective mutants was determined by supplying them with CFA and CMA and by complementation studies with cloned DNA from the COR biosynthetic cluster . Using this approach, the regions encoding CFA and CMA synthesis and coupling activity were localized to the 24-, 12.5- and 2.3-kb regions of the cluster, respectively . Mutants in a 6-kb region required the addition of both CFA and CMA for COR synthesis, which may indicate a regulatory role for this part of the cluster . PS51 and PS61 transconjugants containing cloned DNA from the coupling region produced COR when supplied with CFA and CMA, indicating that coupling activity was cloned and expressed in bacteria lacking the COR biosynthetic cluster.

N Engl J Med, 1993 Oct 28, 329(18), 1308 - 13
Correlation between genotype and phenotype in patients with cystic fibrosis . The Cystic Fibrosis Genotype-Phenotype Consortium; Purification and antipathogenic activity of lipid transfer proteins (LTPs) from the leaves of Arabidopsis and spinach; Laboratorio de Bioquimica y Biologia Molecular, ETS Ingenieros Agronomos, Madrid, SpainTwo homogeneous proteins active in vitro against the bacterial pathogen Clavibacter michiganensis subsp . sepedonicus were obtained from a crude cell-wall preparation from the leaves of Columbia wild-type Arabidopsis . The N-terminal amino acid sequences of these proteins allowed their identification as lipid transfer proteins (LTP-a1, LTP-a2); the LTP1-a1 sequence was identical to that deduced from a previously described cDNA (EMBL M80566) and LTP-a2 was quite divergent (44% identical positions) . These proteins were not detected in the cytoplasmic fraction by Western-blot analysis . Proteins LTP-s1 and LTP-s2 were similarly obtained from spinach leaves; LTP-s1 was 91% identical to a previously purified spinach LTP (Swiss Prot P10976), and LTP-s2 was moderately divergent (71% identical positions) . About 1/3 of the total LTPs were detected in the cytoplasmic fraction from spinach by Western-blot analysis . Concentrations of these proteins causing 50% inhibition (EC-50) were in the 0.1-1 microM range for the bacterial pathogens C . michiganensis and Pseudomonas solanacearum and close to 10 microM for the fungal pathogen Fusarium solani.

J Biol Chem, 1993 Oct 15, 268(29), 21791 - 9
Mechanism of action of Pseudomonas exotoxin . Identification of a rate-limiting step; Zdanovsky AG et al.; Pseudomonas exotoxin (PE) enters cells by receptor-mediated endocytosis and is cleaved by a cellular protease between Arg279 and Gly280 to produce an NH2-terminal fragment of 28 kDa which contains the toxin's binding domain and a COOH-terminal fragment of 37 kDa which has translocating and ADP-ribosylating activity . After proteolysis, the COOH-terminal fragment reaches the endoplasmic reticulum by retrograde transport where it translocates to the cytosol and inhibits protein synthesis by ADP-ribosylating elongation factor 2 . To understand how the 37-kDa fragment functions, we focused on the role of specific amino acids located near its NH2 terminus . We found that there was a 4-250-fold loss in toxic activity when tryptophan 281, leucine 284, or tyrosine 289 were changed to other residues . Mutations at these three positions did not interfere with the receptor binding, cell-mediated proteolytic cleavage, or ADP-ribosylating activity . To determine the role of these amino acids, a competition assay was devised in which the addition of excess PE delta 553, a mutant form of PE that lacks ADP-ribosylation activity, competed efficiently for the toxicity of PE . Excess PE with mutations near the NH2 terminus of the 37-kDa fragment competed poorly . This competition occurred after proteolysis since PEGly276, a mutant form of PE that is not cleaved, did not complete . We conclude that specific amino acids at the NH2 terminus of the 37-kDa fragment interact in a saturable manner with an unknown intracellular component.

J Bacteriol, 1993 Oct, 175(20), 6451 - 8
Genetic organization of a cluster of genes involved in the production of phaseolotoxin, a toxin produced by Pseudomonas syringae pv . phaseolicola; Zhang Y et al.; Phaseolotoxin {N delta(N'-sulfo-diaminophosphinyl)-ornithyl-alanyl- homoarginine} produced by Pseudomonas syringae pv . phaseolicola, the bean halo blight pathogen, is a potent inhibitor of ornithine carbamoyltransferase (OCT) . Inhibition of OCT in infected plants leads to chlorosis and growth inhibition . A genomic cosmid clone, pHK120, containing a 25-kb fragment of DNA from a wild-type strain of P . syringae pv . phaseolicola restores toxin production in Tox- mutants . Tn5 mutagenesis of pHK120 and marker exchange of pHK120::Tn5 plasmids in the wild-type strain resulted in the isolation of 39 chromosomal mutants that harbor Tn5 insertions at known positions . Toxin bioassays revealed that 28 of the mutants, with Tn5 insertions distributed throughout the insert of pHK120, were Tox-, indicating that a functional locus for toxin production in each mutant was inactivated . Complementation analysis was done by testing for toxin production strains that carried a genomic Tn5 at one location and a plasmid-borne Tn5 at another location (pair complementation) . Pair complementation analysis of nine marker exchange mutants and a random genomic Tn5 mutant revealed that there are a minimum of eight toxin loci (phtA through phtH) in pHK120 . Mutants carrying Tn5 insertions in the phtA, phtD, and phtF loci were complemented by deletion subclones containing fragments from pHK120; mutants carrying Tn5 insertions in the phtC locus were partially complemented by a subclone, and mutants carrying Tn5 insertions in the phtB, phtE, phtG, and phtH loci were not complemented by any of the available subclones . A comparison of the insert from pHK120 with that from pRCP17, a clone reported previously (R . C . Peet, P . B . Lindgren, D . K . Wills, and N . J . Panopoulos, J . Bacteriol . 166:1096-1105, 1986) by another laboratory to contain some of the phaseolotoxin genes and the phaseolotoxin-resistant OCT gene, revealed that the inserts in these two cosmids overlap but differ in important respects.

J Bacteriol, 1993 Oct, 175(19), 6169 - 78
vsrB, a regulator of virulence genes of Pseudomonas solanacearum, is homologous to sensors of the two-component regulator family; Huang J et al.; Pseudomonas solanacearum, an important wilt pathogen of many plants, produces several extracellular proteins (EXPs) and extracellular polysaccharides (EPSs) that contribute to its virulence . Using TnphoA mutagenesis, we discovered a new gene, vsrB, that when inactivated causes a major reduction in the virulence and production of an EPS . Analysis of eps::lacZ reporters showed that vsrB is required for maximal expression (transcription) of eps, whose products are required for production of EPS I, a major virulence determinant . Analysis of EXPs in culture supernatants revealed that inactivation of vsrB also causes reduced production of two major EXPs, with molecular masses of 28 and 97 kDa, and a simultaneous 15-fold increase in levels of another EXP, PglA endopolygalacturonase . The vsrB gene was cloned from a P . solanacearum genomic library by complementation of the nonmucoid phenotype of the vsrB::TnphoA mutant and then subcloned on a 2.4-kb DNA fragment . TnphoA fusion analysis and subcellular localization of the vsrB gene product in Escherichia coli maxicells suggest that it is a ca . 60-kDa transmembrane protein . The nucleotide sequence of the 2.4-kb DNA fragment was determined, and a 638-amino-acid open reading frame was found for VsrB . A search of the GenBank data base found that the central part of VsrB has homology with the histidine kinase domain of sensors in the two-component regulator family, while the C terminus has homology with the phosphate receiver domain of response regulators in the same family . Genetic analysis suggests that the receiver domain is not required for vsrB function.

J Bacteriol, 1993 Oct, 175(19), 6075 - 81
Degradation of chloroaromatics: purification and characterization of maleylacetate reductase from Pseudomonas sp . strain B13; Kaschabek SR et al.; Maleylacetate reductase of Pseudomonas sp . strain B13 was purified to homogeneity by chromatography on DEAE-cellulose, Butyl-Sepharose, Blue-Sepharose, and Sephacryl S100 . The final preparation gave a single band by polyacrylamide gel electrophoresis under denaturing conditions and a single symmetrical peak by gel filtration under nondenaturing conditions . The subunit M(r) value was 37,000 (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) . Estimation of the native M(r) value by gel filtration gave a value of 74,000 with a Superose 6 column, indicating that the enzyme is dimeric . The pH and temperature optima were 5.4 and 50 degrees C, respectively . The pI of the enzyme was estimated to be 7.0 . The apparent Km values for maleylacetate and NADH were 58 and 30 microM, respectively, and the maximum velocity was 832 U/mg of protein for maleylacetate . Maleylacetate and various substituted maleylacetates, such as 2-chloro- and 2-methyl-maleylacetate, were reduced at significant rates . NADPH was also used as a cofactor instead of NADH with nearly the same Vmax value, but its Km value was estimated to be 77 microM . Reductase activity was inhibited by a range of thiol-blocking reagents . The absorption spectrum indicated that there was no bound cofactor or prosthetic group in the enzyme.

Cancer Res, 1993 Oct 1, 53(19), 4588 - 94
Polyethylene glycol-modified chimeric toxin composed of transforming growth factor alpha and Pseudomonas exotoxin; Wang QC et al.; Modification of proteins with monomethoxy-polyethylene glycol (mPEG) has been shown to prolong circulation time and to reduce immunogenicity . To make a mPEG-modified recombinant toxin that retained cytotoxic activity but had a longer residence time in circulation, we have constructed an altered form of TGF alpha-PE40, a recombinant toxin composed of human transforming growth factor alpha (TGF alpha) fused to a fragment of Pseudomonas exotoxin (PE38) devoid of its cell-binding domain . In the newly designed protein, termed TGF alpha R29-L2-CH2-PE38QQ delta (TCP), there are no lysine residues in the TGF alpha and PE38 portions . Human IgG4 constant region CH2 and a tetradecapeptide linker; L2, are inserted between TGF alpha and PE38 . Together, L2 and CH2 contain 13 lysine residues as potential modification sites for mPEG . mPEG conjugates of TCP (PEG-TCP) were generated and the products were resolved by ion exchange chromatography . Two PEG-TCP species termed B4 and B6 retained 15 and 4% of cytotoxicity, respectively, and 26% of their receptor binding activity compared with the unmodified TCP . Both B4 and B6 had prolonged circulation times in the blood and reduced toxicity in animals . The mean residence times of B4 and B6 were 37 and 68 min, respectively, compared to 7 min for TCP . When administered i.v . to tumor bearing mice, both B4 and B6 produced marked antitumor effects whereas the unmodified TCP had none . Also, the immunogenicity of PEG-TCP was 5-10 times less than that of TCP . We suggest that the prolonged circulating time and reduced toxicity of PEG-TCP compensate for a diminished cytotoxic activity and enlarge significantly the therapeutic window of this chimeric toxin.

Trends Biochem Sci, 1993 Oct, 18(10), 372 - 6
On the origin of enzymatic species; Petsko GA et al.; The diversity of enzyme catalytic function is remarkable, particularly when one considers that ancestral life forms must have started with a much smaller ensemble of proteins . In this article, we discuss the evolution of the mandelate pathway in pseudomonads as an example of how catalytic diversity may have evolved . We suggest that existing enzymes that catalyse the chemistry needed to accomplish a transformation were recruited, followed by the evolution of specific binding.

Biotechnol Appl Biochem, 1993 Oct, 18 ( Pt 2), 201 - 7
Formate dehydrogenase from methylotrophic bacterium Pseudomonas sp . 101: gene cloning and expression in Escherichia coli; Tishkov VI et al.; Using two degenerate 20- and 23-mer oligonucleotide probes, the gene encoding NAD(+)-dependent formate dehydrogenase (FDH) (EC 1.2.1.2) was shown to lie within a 3.5 kb PstI fragment of the chromosomal DNA of methylotrophic bacterium Pseudomonas sp . 101 . A phasmid library was prepared in the lambda pSL5 vector with partial EcoRI-digested DNA from Pseudomonas sp . 101 . The 12 clones selected contained three types of phasmid: lambda pFDH1, lambda pFDH2 and lambda pFDH3 (with inserts of 15, 17.6 and 18.5 kb respectively) . The inserts contained the same 15 kb EcoRI and 3.5 kb PstI fragments and included the complete FDH gene . Further subcloning of the insert resulted in plasmid pFDH2 and a 2.32 kb HindIII-BgIII fragment . Active enzyme was expressed in Escherichia coli TG1 (pFDH2) strain under control of a lac promoter.

Appl Environ Microbiol, 1993 Oct, 59(10), 3400 - 5
Influence of organic nutrients and cocultures on the competitive behavior of 1,2-dichloroethane-degrading bacteria; van den Wijngaard AJ et al.; The effects of organic nutrients and cocultures on substrate removal by and competitive behavior of 1,2-dichloroethane-degrading bacteria were investigated . Xanthobacter autotrophicus GJ10 needed biotin for optimal growth on 1,2-dichloroethane . In continuous culture, dilution of biotin to a concentration below 0.2 nM resulted in washout . Growth could be restored by inoculation with the 2-chloroethanol utilizer Pseudomonas sp . strain GJ1, leading to a new steady state in which about 1% of the mixed culture consisted of cells of strain GJ1 . This indicates that strain GJ1 excreted biotin or a precursor for its synthesis . Inoculation of the mixed culture with Ancylobacter aquaticus AD25 did not result in washout of strain GJ10, although strain AD25 has a 10-fold-lower Ks for growth on 1,2-dichloroethane . Strain AD25 did not become dominant because of the lack of vitamins, which are necessary for its optimal growth . The results indicate that medium composition and the presence of other species strongly influence the effect of substrate limitation on the composition of a bacterial population that degrades a xenobiotic compound in a continuous culture.

Otolaryngol Clin North Am, 1993 Oct, 26(5), 845 - 56
Biochemical basis of aminoglycoside ototoxicity; Schacht J; The basis for the development of a rational explanation of aminoglycoside toxicity now appears to exist . The acute effects of these drugs are primarily based on calcium antagonism and block of ion channels . The chronic toxicity requires metabolism, and the expression of tissue-specific toxicity is a balance between synthesis of the toxin and its detoxification . Further investigations into the nature of the toxic metabolite should allow us to combine this information with previously established intracellular actions of aminoglycosides to create a unified hypothesis of action . The ability of glutathione to block toxin formation or to increase detoxification (or both) may have clinical implications for the prevention of aminoglycoside-induced ototoxicity . The clinical use of aminoglycosides has somewhat decreased over the last decade because of the introduction of the less toxic cephalosporins of the third generation and the quinolones, which are effective against Pseudomonas infections . Development of bacterial resistance against aminoglycosides is another factor, although resistance to the cephalosporins is also rapidly becoming a serious problem that eventually will limit their usefulness . Only through a detailed knowledge of the molecular basis of toxicity can we rationally pursue the development of new aminoglycosides with less ototoxic and nephrotoxic potential and devise treatments that will prevent the adverse side effects of these antibiotics.

Mol Ecol, 1993 Oct, 2(5), 285 - 93
The cloning and characterization of phage promoters, directing high expression of luciferase in Pseudomonas syringae pv . phaseolicola, allowing single cell and microcolony detection; Waterhouse RN et al.; Regions of DNA containing promoter sequences from a Pseudomonas syringae pv . phaseolicola-specific phage (phi 11P) were identified by shotgun cloning into a broad-host-range promoter-probe vector (pQF70) . When used in conjunction with the luciferase reporter genes, one of these DNA fragments, 19H, directed gene expression at a level which enabled the subsequent light output (bioluminescence) of single cells of P . syringae pv . phaseolicola to be detected and visualized using a charge-coupled device (CCD) . The P . syringae pv . phaseolicola phi 11P, 19H and P . aeruginosa phi PLS27, HcM promoters gave a 50-fold increase in bioluminescence (maximum relative light output) compared to similar constructs containing other well-characterized promoters, for example, tetracycline . Similar bioluminescent characteristics of the transformed bacterium, were observed during growth with and without antibiotic-selection . When lux+ bacteria were inoculated onto French bean leaf (Phaseolus vulgaris L.), the resultant secondary halo blight lesions were bioluminescent and during phylloplane colonization by the lux+ bacterium, bioluminescence on leaf surfaces was detected and imaged by the CCD . Use of these newly identified promoters, combined with the greatly increased sensitivity of bioluminescence detection by the CCD, thus provided a new dimension for the study of natural ecological populations during the bacterial colonization of plants.

Mol Microbiol, 1993 Oct, 10(1), 87 - 97
The major heat-modifiable outer membrane protein CD is highly conserved among strains of Branhamella catarrhalis; Murphy TF et al.; The outer membrane of Branhamella catarrhalis contains a major, heat-modifiable outer membrane protein called CD which has epitopes on the surface of the intact bacterium . The gene encoding CD was cloned and expressed in Escherichia coli . The protein migrates in gels as a doublet, indicating that CD is encoded by single gene whose gene product has two stable conformations . The nucleotide sequence of the gene encoding CD was determined and shows homology with the OprF outer membrane protein of Pseudomonas species . The CD protein contains a proline-rich region, which appears to account for its aberrant migration in gels . Restriction fragment-length analysis of 30 isolates of B . catarrhalis with oligonucleotide probes corresponding to sequences in the CD gene produced identical patterns in Southern blot assays . The major heat-modifiable outer membrane protein CD shares homology with the OprF protein and is highly conserved among strains of B . catarrhalis.

Biosci Biotechnol Biochem, 1993 Oct, 57(10), 1655 - 9
Characterization of the C alpha-dehydrogenase gene involved in the cleavage of beta-aryl ether by Pseudomonas paucimobilis; Masai E et al.; C alpha-dehydrogenase catalyzes the oxidation of arylglycerol-beta-aryl ether at the C alpha-position, and therefore this process produces the specific substrate for beta-etherase, which cleaves beta-ary ethers (C alpha carbonyl type) . Here we isolated the C alpha-dehydrogenase gene (lig D) and sequenced its nucleotides . This gene contains an open reading frame of 915 bp and the deduced amino acid sequence had a homology with the ribitol dehydrogenase family . lig D is about 1 kbp upstream of the beta-etherase gene (lig E).

Infect Control Hosp Epidemiol, 1993 Oct, 14(10), 583 - 6
Nosocomial outbreak of Pseudomonas cepacia associated with contamination of reusable electronic ventilator temperature probes; Weems JJ Jr; OBJECTIVE: The investigation and control of an outbreak of nosocomial Pseudomonas cepacia respiratory tract colonization and infection . DESIGN: Epidemiologic investigation based on infection control surveillance data, including definition and characterization of case patients, environmental cultures based on epidemiologic information, and institution of control measures based on study results . SETTING: A 1,171-bed, university-affiliated tertiary care hospital . RESULTS: Between January 1, 1988, and June 30, 1989, 127 patients were culture-positive for P cepacia, 117 (92%) of whom were culture-positive from sputum and were treated in the intensive care unit . Review of respiratory care procedures revealed that when mechanical ventilators were serviced between patients, the electronic temperature probes used with servo-controlled humidifiers were wiped with the same odor-counteractant cleaning solution used on ventilator cabinets . P cepacia was isolated from nine of 12 in-use temperature probe tips, three of which were from patients with negative sputum cultures for P cepacia, one of whom subsequently developed culture positivity for P cepacia . P cepacia also was isolated from the diluted odor-counteractant solution . Following the institution of a disinfection procedure for temperature probe tips, the incidence of P cepacia sputum culture positivity in ICU patients fell significantly compared to the outbreak period (138 of 5,225 discharges versus 52 of 3,678 discharges, P < 0.01) . CONCLUSIONS: This investigation identified contamination of reusable electronic temperature probes as a source of nosocomial respiratory infection due to P cepacia and emphasizes the need to carefully evaluate disinfection practices for reusable patient care equipment.

J Neurosurg, 1993 Oct, 79(4), 569 - 76
Cytotoxicity and antitumor effects of growth factor-toxin fusion proteins on human glioblastoma multiforme cells; Kunwar S et al.; The prognosis of glioblastoma multiforme remains poor despite advances in treatment by surgery, irradiation, and chemotherapy . Many malignant gliomas overexpress growth factor receptors . The possibility of targeting these receptors with selective cytotoxic molecules constructed by fusing deoxyribonucleic acid (DNA)-encoding mutant forms of Pseudomonas exotoxin A (PE) with complementary DNA-encoding growth factors was investigated . Several recombinant toxins have been produced, including those in which transforming growth factor (TGF)-alpha, insulin-like growth factor (IGF)-I, and acidic fibroblast growth factor (FGF) were fused to mutant forms of PE lacking the native cell-binding domain . These recombinant proteins are cytotoxic to cells that express specific cell-surface receptors . The cytotoxic activity of TGF-alpha, IGF-I, and acidic FGF chimeric toxins was tested in vitro against human glioblastoma cell lines . Each recombinant toxin exhibited potent and specific killing of cells . The TGF-alpha-PE40 construct was cytotoxic to seven of the eight cell lines and was active at concentrations as low as 0.5 ng/ml (1.1 x 10(-11) M) . The acidic FGF-PE40 toxin was also active on seven of the eight cell lines but was 50-fold less active than the TGF-alpha-PE40 . The IGF-I-PE40 construct was active on only two cell lines . To determine the possible therapeutic effect in animals, TGF-alpha-PE40 was administered to nude mice bearing subcutaneous human glioblastoma xenografts . The animals were treated for 7 days via a continuous infusion pump placed in the peritoneal cavity . A constant serum level of TGF-alpha-PE40 was achieved that was nontoxic to the mice yet caused a reduction in tumor volume and retarded growth beyond the treatment period . The overexpression of the epidermal growth factor receptor in glioblastomas multiforme and the potency and specificity of the TGF-alpha-PE40 construct designed to target this receptor suggests that TGF-alpha-PE40 has the potential to be an effective antitumor agent for the adjuvant therapy of these carcinomas.

Infect Immun, 1993 Oct, 61(10), 4045 - 50
Interaction of insulin with Pseudomonas pseudomallei; Woods DE et al.; Pseudomonas pseudomallei is the causative agent of melioidosis, a disease being increasingly recognized as an important cause of morbidity and mortality in many regions of the world . An intriguing observation regarding melioidosis is that a significant percentage of patients who develop the disease have preexisting diabetes mellitus . In this regard, we have tested the hypothesis that insulin may modulate the growth of P . pseudomallei . We have demonstrated that insulin markedly inhibits the growth of P . pseudomallei in vitro and in vivo . The growth rate of P . pseudomallei in minimal medium containing human recombinant insulin was significantly lower than that of control cultures containing no insulin . P . pseudomallei grew at an increased rate in serum samples obtained from diabetic rats compared with that in serum samples obtained from control animals . When the insulin level was restored by the addition of human recombinant insulin, the growth rate was reduced to a level similar to that seen in control serum . P . pseudomallei also grew significantly better in insulin-depleted human serum than control human serum . 125I-insulin binding studies demonstrated that P . pseudomallei possesses a specific, high-affinity binding site for human insulin . In in vivo studies, rats made diabetic by streptozotocin injection (80 mg/kg of body weight, intraperitoneally) were significantly more susceptible to P . pseudomallei septicemia than control rats . Thus, it appears that serum insulin levels may play a significant role in modulating the pathogenesis of P . pseudomallei septicemic infections.

Am J Respir Cell Mol Biol, 1993 Oct, 9(4), 455 - 62
Release of interleukin-8, interleukin-6, and colony-stimulating factors by upper airway epithelial cells: implications for cystic fibrosis; Bedard M et al.; Cystic fibrosis (CF) is characterized by a dramatic neutrophil recruitment and repeated Pseudomonas infections in the lungs . To evaluate cytokine releasibility by airway epithelial cells in the context of CF, we studied primary nasal epithelial cells isolated from the upper airways and continuous epithelial cell lines from normal and CF subjects . Relatively low levels of interleukin (IL)-8, IL-6, and granulocyte/macrophage colony-stimulating factor (GM-CSF) were produced spontaneously by primary epithelial cells (< 50 pg/10(6) cells) and higher levels of colony-stimulating factor-1 (CSF-1) (1 to 2 ng/10(6) cells) . Cells were stimulated with substances that are likely to be present in the inflamed lungs of CF patients-namely, the proinflammatory monokines IL-1 and tumor necrosis factor-alpha (TNF alpha) as well as neutrophil elastase and bacterial products from Pseudomonas (mucoid exopolysaccharide {MEP} and rhamnolipids) . Both IL-1 and TNF alpha induced a dose-dependent release of IL-6 (5 to 10 ng/10(6) cells) and GM-CSF (2 to 3 ng/10(6) cells) by primary epithelial cells from eight normal volunteers . The TNF alpha/IL-1-stimulated GM-CSF release was blocked by the addition of 1 microM dexamethasone, whereas basal CSF-1 release was unaffected . Neutrophil elastase was a potent inducer of IL-8 and GM-CSF both in primary epithelial cells and in cell lines . Dexamethasone (1 microM) did not inhibit elastase-induced IL-8 release in either normal or CF epithelial cells . Rhamnolipids and MEP were found to stimulate the copious release of IL-8, GM-CSF, and IL-6 from epithelial cells, in a steroid-sensitive fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Surg Oncol, 1993 Oct, 2(5), 293 - 8
Combined percutaneous transhepatic and endoscopic placement of biliary stents; Hunt JB et al.; Combined percutaneous transhepatic cholangiography and endoscopic retrograde cholangiography can be used to stent biliary obstruction when attempts at endoscopic stenting have failed . Between January 1987 and August 1991 we performed 35 combined procedures in 31 patients with malignant obstruction . Post stenting serum bilirubin and serum alkaline phosphatase concentration fell after 33 and 29 procedures, respectively . In six studies there was evidence of infection prior to stenting . In spite of the use of prophylactic antibiotics, septic complications developed after eight procedures (23%) . Pseudomonas was the most commonly isolated pathogen (46%) . Twenty-three patients were discharged, 30-day mortality was 8 (23%) and median survival was 14 weeks (range 0-75 weeks) . Seven patients required eight stent changes because of blockage (median patency time 18 weeks; range 7-75 weeks) . Use of this technique allows relief of biliary obstruction . Potential infective and bleeding complications must be anticipated.

J Clin Microbiol, 1993 Oct, 31(10), 2589 - 93
Arbitrarily primed polymerase chain reaction as a rapid method to differentiate crossed from independent Pseudomonas cepacia infections in cystic fibrosis patients; Bingen EH et al.; We used DNA fingerprinting by the arbitrarily primed polymerase chain reaction (AP-PCR) technique for an epidemiological investigation of 23 Pseudomonas cepacia isolates obtained from 11 cystic fibrosis (CF) patients attending our CF center . This approach was compared with ribotyping, pulsed-field gel electrophoresis (PFGE), and conventional phenotypic typing . AP-PCR and ribotyping were identical in resolving power, since the two methods generated four different profiles and identified the same group of strains . Six patients on the one hand and four on the other harbored strains of the same genotype, thus raising the possibility of either patient-to-patient transmission or acquisition from a common hospital environmental source . PFGE results were in good agreement with those of the other two methods, but PFGE seems more discriminative since it generated a fifth profile for a single strain in a group of four . Our results show in vivo stability for the three methods during a period extending from 3 to 41 months . These genotypic techniques are particularly promising for clinical laboratories to help to clarify the epidemiology of P . cepacia in CF patients . The AP-PCR method constitutes an easier alternative to the well-established ribotyping method . AP-PCR provides the quickest results with minimal technical complexity . However, our results suggest that it is less discriminative than the labor-intensive PFGE method.

FEBS Lett, 1993 Sep 27, 331(1-2), 123 - 8
The crystal structure of triacylglycerol lipase from Pseudomonas glumae reveals a partially redundant catalytic aspartate; Noble ME et al.; The family of lipases (triacylglycerol-acyl-hydrolases EC 3.1.1.3) constitutes an interesting class of enzymes because of their ability to interact with lipid-water interfaces, their wide range of substrate specificities, and their potential industrial applications . Here we report the first crystal structure of a bacterial lipase, from Pseudomonas glumae . The structure is formed from three domains, the largest of which contains a subset of the alpha/beta hydrolase fold and a calcium site . Asp263, the acidic residue in the catalytic triad, has previously been mutated into an alanine with only a modest reduction in activity.

Biochemistry, 1993 Sep 7, 32(35), 9148 - 55
Structural characterization of the divalent cation sites of bacterial phosphotriesterase by 113Cd NMR spectroscopy; Omburo GA et al.; The phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of organophosphate esters . The isolated native protein contains zinc, and removal of this metal abolishes the enzymatic activity . Reconstitution of the apoenzyme requires 2 mol of cadmium per mol of protein for full catalytic activity . The kcat and Km values for the hydrolysis of paraoxon for the cadmium-substituted enzyme are 4300 s-1 and 390 microM, respectively . These values compare favorably with the kinetic constants observed for the zinc-substituted enzyme (2300 s-1 and 78 microM) . A hybrid enzyme containing one zinc and one cadmium ion is catalytically active, and the kinetic constants are nearly identical to the values obtained with the all-zinc-containing enzyme . The NMR spectrum of protein reconstituted with two 113Cd2+ ions per enzyme molecule exhibits cadmium resonances at 212 and 116 ppm downfield from Cd(ClO4)2 . The two metal ions are, therefore, in significantly different chemical environments . These two binding sites have been designated the M alpha and M beta sites for the low- and high-field signals, respectively . Protein substituted with a single cadmium ion also shows two cadmium resonances, and thus one site is not completely filled prior to the binding of metal to the other site . The Cd/Zn hybrid protein shows a single cadmium resonance at 115 ppm, and thus the cadmium is occupying the M beta site while zinc is occupying the M alpha site . The positions of the observed chemical shifts for the two cadmium signals indicate that the ligands to both metals are composed of a mixture of oxygen and nitrogen atoms.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1993 Sep, 175(18), 6028 - 37
Cloning and expression of a member of a new cytochrome P-450 family: cytochrome P-450lin (CYP111) from Pseudomonas incognita; Ropp JD et al.; Cytochrome P-450lin catalyzes the 8-methyl hydroxylation of linalool as the first committed step of its utilization by Pseudomonas incognita as the sole carbon source . By using a polymerase chain reaction-based cloning strategy, a 2.1-kb DNA fragment containing the cytochrome P-450lin gene (linC) was isolated . An open reading frame of 406 amino acids has been identified as that of P-450lin on the basis of amino acid sequence data from peptides of the native protein . Heterologous expression of functional holoprotein is exhibited by Escherichia coli transformed with pUC18 containing the subcloned linC gene under constitutive transcriptional control of the lac promoter . The G+C content of linC was found to be 55% overall and 58% in the third codon position . An optimized amino acid sequence alignment of P-450lin with cytochrome P-450cam shows that the two enzymes have only 25% identity . P-450lin was found to exhibit the expected conservation in the axial cysteine heme ligand-containing peptide and the threonine region postulated to form an O2-binding pocket (T . L . Poulos, B . C . Finzel, and A . J . Howard, J . Mol . Biol . 195:687-700, 1987) . The low amino acid sequence identity between P-450lin and all other P-450 sequences has shown that P-450lin is the first member of the CYP111 P-450 gene family.

J Bacteriol, 1993 Sep, 175(18), 5916 - 24
Characterization of the promoter of avirulence gene D from Pseudomonas syringae pv . tomato; Shen H et al.; The avirulence gene D (avrD) from Pseudomonas syringae pv . tomato comprises the first open reading frame (ORF) of a putative operon consisting of at least five tandem ORFs . The promoter of the avrD operon was localized to a 150-bp DNA fragment occurring 5' to the avrD gene by using the Tn7-lux and gus reporter systems . The avrD promoter in P . syringae pv . tomato and P . syringae pv . glycinea was poorly expressed when bacteria were grown in complex culture media but was activated during bacterial growth in plants . The timing and level of induction were similar in compatible and incompatible plant-pathogen interactions . When bacteria were grown in minimal culture medium, promoter activity was repressed by certain carbon sources, high concentrations of nitrogen compounds, and pH values above 6.5 . Primer extension experiments on RNA from bacteria grown in minimal medium identified two transcription initiation sites 87 and 41 nucleotides upstream from the translational start site . Only the -41 transcriptional start site was identified in bacteria grown in soybean leaves . A sigma 54 promoter consensus sequence (GG-10 bp-GC) occurred 14 bp upstream of the -41 transcriptional start, and 3' deletions into this region completely abolished promoter activity . Little expression was observed when a gus fusion with the avrD promoter was introduced into an ntrA mutant strain of P . syringae pv . phaseolicola deficient in the sigma 54 cofactor . Expression from the avrD promoter also required the hrp regulatory genes, hrpS and hrpL . Deletions from the 5' end of the promoter region and base substitution analyses also identified two upstream elements important for expression . Sequence comparison of these elements with other cloned avirulence genes revealed the presence of a conserved consensus sequence elements with other cloned avirulence genes revealed the presence of a conserved consensus sequence (GGAACC-N15/16-CCAC) in the promoters of nine different avirulence genes from P . syringae pathovers.

Am J Surg, 1993 Sep, 166(3), 284 - 8
Immunotoxins and recombinant toxins in the treatment of solid carcinomas; Theuer CP et al.; Cancer remains the second most common cause of death in our society, and advanced disease is often refractory to surgical, chemotherapeutic, and radiologic interventions . One novel approach to cancer treatment involves targeting a cytotoxic agent to a cancer cell . Immunotoxins have been developed that contain a potent toxin (either Pseudomonas exotoxin, ricin toxin, or diphtheria toxin) coupled to a targeting moiety that directs the molecule to cells expressing a certain antigen . Chemically coupled immunotoxins have been developed over the past 12 years . These bind to and kill cells expressing many tumor-associated antigens . Initial clinical results were disappointing, but recent results have been more promising . Furthermore, newer immunotoxins have been developed that will soon be in clinical trials . Some of these are recombinant toxins that have been developed using techniques of genetic engineering . Transforming growth factor-alpha, acidic fibroblast growth factor, insulin-like growth factor-1, interleukin-2, interleukin-4, interleukin-6, the binding portions of monoclonal antibodies, and CD4 have been used to direct toxins to cancer cells or cells infected with the human immunodeficiency virus type 1 . Efforts are under way to circumvent problems such as immunogenicity that may limit the clinical usefulness of immunotoxins.

J Bacteriol, 1993 Sep, 175(17), 5477 - 87
Phenotype conversion in Pseudomonas solanacearum due to spontaneous inactivation of PhcA, a putative LysR transcriptional regulator; Brumbley SM et al.; Phenotype conversion (PC) in Pseudomonas solanacearum is the coordinated change in production of extracellular polysaccharide and a variety of extracellular proteins, some of which contribute to virulence . Although PC is normally spontaneous, it is mimicked by transposon inactivation of the phcA locus (S . M . Brumbley and T . P . Denny, J . Bacteriol . 172:5677-5685, 1990) . The DNA sequence of a 1.8-kb region from strain AW1 that contains phcA revealed one open reading frame that should encode a polypeptide of 38.6 kDa . The PhcA protein produced in Escherichia coli by using a T7 RNA polymerase expression system was of the predicted size . The deduced amino acid sequence of PhcA is similar to that of some members of the LysR transcriptional activator gene family, especially in the amino terminus, where a putative helix-turn-helix DNA-binding motif was identified . An analogous allele (phcA1) was cloned from the spontaneous PC mutant strain AW1-PC and found to be nonfunctional in complementation studies . When phcA1 was expressed in E . coli, the PhcA1 protein was 35.5 kDa, 3 kDa smaller than PhcA . Sequence analysis of phcA1 and chimeric constructs of phcA and phcA1 confirmed that PhcA1 is truncated by a 2-bp insertion 147 nucleotides upstream of the carboxyl terminus of PhcA . Southern blot analysis of 10 additional independently isolated PC mutants of strain AW1 revealed that two strains have larger insertions (0.2 and 1.0 kb) within phcA . These results suggest that phcA encodes a DNA-binding protein that regulates the transcription of one or more of the genes involved in P . solanacearum virulence and that spontaneous PC can be attributed to one of several different insertions within this locus.

Chest, 1993 Sep, 104(3), 681 - 5
Infection in the transplanted and native lung after single lung transplantation; Horvath J et al.; OBJECTIVE: To analyze a single-center experience with infectious complications of single lung transplantation (SLT) with special emphasis on risk factors for infection in the transplanted and native lung . DESIGN: Consecutive case series . SETTING: University teaching hospital . PATIENTS: Fifteen consecutive SLT recipients (mean age, 43 years; 9 men and 6 women) . Mean follow-up was 337 days . RESULTS: Fifteen patients had 24 infectious episodes (1.6 per patient) of which 83 percent were life-threatening, 79 percent involved the lung, airway, or pleural space, and 79 percent occurred in the first 4 months after transplantation . Despite this high infectious morbidity, there were no infectious deaths . The most important infections were bacterial pneumonia (n = 10), cytomegalovirus (CMV) pneumonia (n = 5), and bronchial anastomotic infections (n = 3) . Significant risk factors for bacterial pneumonia were a diagnosis of primary or secondary pulmonary hypertension (p < 0.05) and the presence of airway complications of stenosis or dehiscence (p < 0.05) . No risk factors for overall lung infections were identified . The native lung was involved in 6 of 16 lung infections and was the exclusive site of infection in 4 cases . Underlying disease in the native lung may have predisposed to infection at that site by a mechanism of inadequate blood flow or impaired ventilation . Three bronchial anastomotic infections (Pseudomonas, Candida, Aspergillus) occurred, all with dehiscence of the anastomosis . These were highly morbid but resolved with antibiotics, stent placement, and surgical retention in two of the three cases . The five episodes of CMV pneumonia caused mild (four patients) or moderate (one patient) dysfunction and responded to antiviral agents without relapse . CONCLUSION: The frequency, complexity, and morbidity of infections after SLT were great, but most infections were manageable and good outcomes were achieved . A pretransplant diagnosis of pulmonary hypertension or posttransplant occurrence of bronchial stenosis or dehiscence were associated with a higher rate of bacterial pneumonia . The underlying disease in the native lung may predispose to infection at that site.

Cardiovasc Res, 1993 Sep, 27(9), 1691 - 7
Cytotoxic effects of vascular smooth muscle cells of the chimeric toxin, heparin binding TGF alpha-Pseudomonas exotoxin; Fu YM et al.; OBJECTIVE: Smooth muscle cell proliferation appears to be very important in restenosis after angioplasty . A chimeric toxin created by genetically fusing the gene encoding TGF alpha (targets the EGF receptor) to the gene encoding Pseudomonas exotoxin (PE) preferentially kills rapidly proliferating smooth muscle cells . Recently, a heparin binding EGF-like growth factor (HB-EGF) has been identified . The HB domain enhances the mitogenic activity for smooth muscle cells . The purpose of this study was to design a new chimeric toxin, having both heparin binding and EGF receptor binding function, and to determine whether it is more cytotoxic to smooth muscle cells . METHODS: By recombinant DNA techniques, a new chimeric toxin, HB-TGF alpha-PE4EKDEL, was synthesised . Cytotoxic assays were performed by assessing the capacity to inhibit protein synthesis of rat vascular smooth muscle cells . RESULTS: The toxin preferentially killed rapidly proliferating smooth muscle cells (p < 0.025) . The HB domain increased the cytotoxicity of the molecule when compared to the other chimeric toxins tested against smooth muscle cells . The cytotoxic effect of the new molecule was significantly decreased by exogenously added heparin (p < 0.05) . CONCLUSIONS: The presence of a heparin binding domain increases the smooth muscle cell cytotoxicity of the TGF alpha fusion toxin, perhaps because HB-TGF alpha-PE4EKDEL functions as a molecule with two ligands . It will be important to determine whether the greater smooth muscle cell cytotoxicity that exists in vitro will facilitate the specific targeting and killing of rapidly proliferating cells in vivo.

Mol Plant Microbe Interact, 1993 Sep-Oct, 6(5), 582 - 91
Recognition of the avirulence gene avrB from Pseudomonas syringae pv . glycinea by Arabidopsis thaliana; Wanner LA et al.; The response of Arabidopsis thaliana land race Columbia to the bacterial pathogen Pseudomonas syringae pv . maculicola 4326 harboring cloned avirulence genes avrB and avrC from P . syringae pv . glycinea and avrA and avrD from P . syringae pv . tomato was examined . Only avrB was recognized by Columbia, as evidenced by attenuation of disease symptoms, slower bacterial multiplication in planta, and differentially greater induction of mRNA for several defense-related genes . This contrasts with two A . thaliana land races where P . s . pv . maculicola strains containing avrB were not recognized . These plants showed typical disease symptoms, and bacterial multiplication in planta was not reduced in response to P . s . pv . maculicola containing avrB . In addition, there was no differential induction of defense-related mRNAs in these susceptible land races after infiltration with bacteria containing or lacking avrB . These results extend previous observations that avirulence genes from pathogens of one host plant can be recognized by "nonhost" plants and provide the genetic framework for analysis of the plant-specified response to the bacterial avrB gene product in A . thaliana.

J Gen Microbiol, 1993 Sep, 139 ( Pt 9), 1967 - 72
A novel pathway for the catabolism of 4-nitrotoluene by Pseudomonas; Rhys-Williams W et al.; Eleven strains of Pseudomonas were isolated by selective enrichment on 4-nitrotoluene (4NT) . They all utilized 4NT, 4-nitrobenzyl alcohol (4NBA) or 4-nitrobenzoate (4NBZate) as sole sources of carbon and nitrogen . One strain, TW3, was used for more detailed studies . 4NT-grown cells of TW3 take up O2 when incubated in the presence of 4NBA, 4-nitrobenzaldehyde (4NBZ) and 4NBZate . HPLC analysis of culture supernatants showed that 4NBZ and 4NBZate were formed when 4NT-grown cells wer incubated with 4NBA, whereas only 4NBZate was found when they were incubated with 4NBZ . Two dehydrogenases were detected in extracts of 4NT-grown cells . 4NBA dehydrogenase could be assayed by a dye-linked assay whereas 4NBZ dehydrogenase activity was linked to NAD+ reduction . No nitrite was detected in supernatants of 4NBZate-grown cells incubated with 4NBZate but the nitrogen appeared as ammonium . The only aromatic ring-cleavage dioxygenase that was induced during growth on the nitroaromatics was protocatechuate 3,4-dioxygenase . It is proposed that the pathway for 4NT catabolism proceeds via 4NBA, 4NBZ and 4NBZate and ultimately to protocatechuate with release of the nitro group as ammonium.

J Clin Immunol, 1993 Sep, 13(5), 359 - 70
Anti-Pseudomonas aeruginosa IgG subclass titers in patients with cystic fibrosis: correlations with pulmonary function, neutrophil chemotaxis, and phagocytosis; Cowan RG et al.; To explore possible mechanisms for the association between elevated immunoglobulin levels and lower pulmonary function in cystic fibrosis patients, we measured serum IgG subclass levels and anti-P . aeruginosa IgG subclass titers and correlated levels with neutrophil phagocytosis and chemotaxis . Serum was obtained from 13 cystic fibrosis patients colonized with the same serotype of P . aeruginosa, 12 noncolonized patients, and 12 normal volunteers . All anti-P . aeruginosa IgG subclass titers were elevated in serum from colonized patients . IgG3 level and anti-P . aeruginosa IgG3 titer were inversely correlated with pulmonary function . Phagocytosis of P . aeruginosa by neutrophils correlated with serum IgG3 level and was increased by opsonization with serum from colonized patients . Chemotactic index was increased in serum from colonized patients and inversely correlated with pulmonary function chest roentgenogram score . Chemotactic index directly correlated with anti-P . aeruginosa IgG3 titer and serum IgG3 . These data demonstrate that cystic fibrosis patients with increased IgG3 levels are in poorer clinical condition and that their serum enhances neutrophil function . Such patients may have increased pulmonary inflammation with subsequent lung damage.

Appl Environ Microbiol, 1993 Sep, 59(9), 2963 - 8
NADP(+)-dependent D-threonine dehydrogenase from Pseudomonas cruciviae IFO 12047; Misono H et al.; NADP(+)-dependent D-threonine dehydrogenase (EC 1.1.1.-), which catalyzes the oxidation of the 3-hydroxyl group of D-threonine, was purified to homogeneity from a crude extract of Pseudomonas cruciviae IFO 12047 . The enzyme had a molecular mass of about 60,000 Da and consisted of two identical subunits . In addition to D-threonine, D-threo-3-phenylserine, D-threo-3-thienylserine, and D-threo-3-hydroxynorvaline were also substrates . However, the other isomers of threonine and 3-phenylserine were inert . The enzyme showed maximal activity at pH 10.5 for the oxidation of D-threonine . The enzyme required NADP+ . NAD+ showed only slight activity . The enzyme was not inhibited by EDTA, o-phenanthroline, alpha,alpha'-dipyridyl, HgCl2, or p-chloromercuribenzoate but was inhibited by tartronate, malonate, pyruvate, and DL-2-hydroxybutyrate . The inhibition by these organic acids was competitive against D-threonine . Initial-velocity and product inhibition studies suggested that the oxidation proceeded through a sequential ordered Bi Bi mechanism . The Michaelis constants for D-threonine and NADP+ were 13 and 0.12 mM, respectively.

Am J Physiol, 1993 Sep, 265(3 Pt 2), H943 - 8
Nitric oxide does not mediate the attenuated pulmonary vascular reactivity of chronic pneumonia; Yaghi A et al.; Chronic Pseudomonas pneumonia is associated with decreased acute hypoxic pulmonary vasoconstriction . However, it is not known whether this is a result of a generalized reduction in contractile responsiveness . We therefore examined the effect of chronic Pseudomonas pneumonia on in vitro pulmonary vascular responsiveness to agonists . We then investigated the role of nitric oxide (NO) in the altered pulmonary vascular contractility . Control rats or rats infected with Pseudomonas (pneumonia) were killed, and small intrapulmonary arteries (100-200 microns effective lumen radius) were removed . In the pneumonia group, arteries were harvested from the pneumonic area of the lung . Vascular responsiveness was assessed in vitro by obtaining cumulative dose-response curves to contractile agonists {phenylephrine (PE), 5-hydroxytryptamine (5-HT), prostaglandin F2 alpha (PGF2 alpha), and KCl} . KCL-induced (voltage-operated) contractions were not significantly depressed in small pulmonary arteries from pneumonic lungs, suggesting that the smooth muscle contractile apparatus in these arteries was preserved . Contractile responses to the three receptor-operated agonists (PE, 5-HT, and PGF2 alpha) were significantly depressed in arteries subserving the pneumonic lobe of infected rats . NG-monomethyl-L-arginine, which blocks the synthesis of NO, caused a shift toward the left in the dose-contraction curves to PE, PGF2 alpha, and 5-HT in vessels from the sterile control lungs, but it had little effect on arteries from the pneumonic lungs . Chronic Pseudomonas pneumonia is associated with depressed pulmonary vascular contractility in vitro, particularly affecting the receptor-mediated contractile responses . Excessive NO release does not contribute to this attenuated vascular contractility.(ABSTRACT TRUNCATED AT 250 WORDS)

Shi Yan Sheng Wu Xue Bao, 1993 Sep, 26(3), 289 - 95
{Gene cloning, expression of fusion protein TGF alpha-PE 40 and its inhibition activity on cancer cell growth}; Xu YH et al.; Recombinant plasmid pYX382 and pYX3825 were constructed by fusing the cDNA encoding transforming growth factor type alpha (TGF alpha) to Pseudomonas exotoxin gene (PE) in which the cell recognition domain was deleted . The chimeric proteins produced by host E . Coli cells BL21 transformed by plasmid pYX382 and pYX3825 are termed TGF alpha-PE 40 which reacts with antibody against TGF alpha or antibody against PE in immunoblotting to show a 46 kd protein band reflecting the fusion of 56 kD TGF alpha peptide and 40 kD truncated Pseudomonas exotoxin molecule . An additional signal sequence OmpA was inserted into upstream region of TGF alpha cDNA in plasmid pYX3825 resulting in the partly secreting of expression product into medium and periplasm of the cells . TGF alpha-PE 40 was purified from medium by MONO Q ion exchange column and TSK 250 gel filtration column attached to Pharmacia EPLC system . The TGF alpha-PE 40 molecules showed a very strong activities inhibiting the protein synthesis and killing the cancer cells overexpressing EGF receptor on the cell surface.

Southeast Asian J Trop Med Public Health, 1993 Sep, 24(3), 436 - 43
The 1990-1991 outbreak of melioidosis in the Northern Territory of Australia: clinical aspects; Currie B et al.; From November 1990 to June 1991, 33 cases of acute melioidosis were diagnosed in tropical Northern Territory, Australia during an exceptionally wet monsoon . Eighteen (55%) were alcoholic, 16 (48%) diabetic and only 4 (12%, all survivors) had no risk factors . Twenty-seven (82%) were considered recent infection, with an incubation period of 3-21 days (mean 14) documented in eight cases with presumed cutaneous inoculation . Fourteen patients presented with pneumonia (4 septicemic) and of 11 others with septicemia 4 had genitourinary foci . Three of 4 with splenic abscesses required splenectomy . Three had only skin/soft tissue infection . One patient with brainstem encephalitis needed prolonged ventilation . Overall mortality was 36% (12 cases, including three relapses), despite therapy with ceftazidime and intensive care facilities . Pseudomonas pseudomallei is the commonest diagnosed cause of fatal bacteremic pneumonia at Royal Darwin Hospital and emphasis is placed on early appropriate antibiotic therapy and compliance with maintenance therapy for at least three months.

Semin Respir Infect, 1993 Sep, 8(3), 207 - 15
Infection after lung transplantation; Paradis IL et al.; Infections have been and still are the major cause of morbidity and mortality after lung transplantation . Nevertheless, the negative impact of infection on outcome has lessened considerably over the last decade because of lessons learned in the prevention, identification, and treatment of infection . Antibiotics tailored to the results of cultures and stains of respiratory-tract secretions obtained from both the donor and recipient have markedly decreased the prevalence of bacterial pneumonia early after lung transplantation . Ganciclovir treatment has reduced the mortality of cytomegalovirus (CMV) disease from 27% to 1% . Ganciclovir as prophylaxis has modestly reduced the prevalence of CMV illness from 80% to 60% . Pneumocystis infection has been nearly eliminated with low-dose trimethropin/sulfamethoxazole prophylaxis . Treating all Candida/Aspergillus isolates from respiratory-tract secretions with fluconazole or itraconazole has reduced the prevalence of fungus infections from 14% to 5% . Challenges still remain . The ideal regimen to prevent CMV illness is yet to be determined, and treating all fungal isolates from the allograft is not cost effective . Recurrent airway infection and late bacterial pneumonia caused by Pseudomonas species when obliterative bronchiolitis is present remain a major cause of concern . Surely the next decade will provide new insights into these problems.

J Hosp Infect, 1993 Sep, 25(1), 33 - 43
Ventilator temperature sensors: an unusual source of Pseudomonas cepacia in nosocomial infection; Berthelot P et al.; A prospective study was undertaken to determine the source of Pseudomonas cepacia colonization and infection that had affected ventilated patients in an Intensive Care Unit (ICU) for three years . Thirty-eight patients undergoing mechanical ventilation were enrolled during a six-week period . Samples were taken from patients, ventilator circuits and the environment for culture . P . cepacia was isolated from the condensate formed in the ventilator circuit and the source of the contamination was shown to be the temperature sensor . Ribotyping of the representative strains of P . cepacia performed with two endonucleases, EcoRI and PvuII, confirmed the homogeneity of the isolates from patients and ventilator circuits . A modification of the procedure for disinfection of the temperature sensors resulted in the eradication of P . cepacia from the ICU.

Kansenshogaku Zasshi, 1993 Sep, 67(9), 816 - 22
Experimental pneumonia produced by clinical isolates of Pseudomonas cepacia in mice; Yamagishi Y et al.; An experimental Pseudomonas cepacia lung infection was induced in ddY mice pretreated with cyclophosphamide . A single dose of 250 mg of cyclophosphamide per kg resulted in leukopenia which lasted for four days . At the lowest PMN levels, the mice were exposed to various doses of bacteria by either intratracheal inoculation or aerosol inhalation . Experimental pneumonia was established by intratracheal inoculation of 1 x 10(7) - 2 x 10(8) colony-forming units of P . cepacia . The duration of survival time of the mice and the number of viable bacteria in their lungs were determined . A dramatic rise in the viable counts of P . cepacia was found within 24 hours after intratracheal inoculation of 1 x 10(8) colony-forming units of P . cepacia . It was impossible to establish P . cepacia pneumonia by aerosol inhalation, since the bacteria were immediately cleared from the lung . Mice which had not been treated with cyclophosphamide remained healthy and did not show any lung lesions . Thus, neutrophils appear to play an important role in the early defense mechanisms of the lung against P . cepacia . This animal model could be of use in evaluating additional therapies for the infection, and the pathologic determinants of infection caused by P . cepacia.

Biochem Cell Biol, 1993 Sep-Oct, 71(9-10), 417 - 20
Structure of the lipopolysaccharide O-antigen of Pseudomonas cepacia serotype A; Beynon LM et al.; The O-antigenic polysaccharides of the lipopolysaccharides produced by three isolates of Pseudomonas cepacia serogroup A were analysed by composition, methylation, periodate oxidation, and two-dimensional nuclear magnetic resonance methods . They were found to be identical linear unbranched polymers of a repeating trisaccharide unit containing 2-acetamido-2-deoxy-D-galactose and L-rhamnose residues (2:1) and have the structure {-->3)-alpha-D-GalpNAc-(1-->3)-beta-D-GalpNAc-(1-->4)-alpha-L-Rhap -(1-->}n.

Southeast Asian J Trop Med Public Health, 1993 Sep, 24(3), 425 - 35
The 1990-1991 outbreak of melioidosis in the Northern Territory of Australia: epidemiology and environmental studies; Merianos A et al.; From November 1990 to June 1991 33 acute cases of melioidosis occurred in the Northern Territory, Australia; 25 cases were reported in the capital city, Darwin . We carried out an epidemiological investigation to exclude a common source outbreak, describe the risk factors for disease, and develop and institute appropriate control measures . We compared population based attack rates among various risk groups using logistic regression, and the demographic, medical and behavioral risk factors for melioidosis by a matched case-control study . Environmental Health Officers collected soil, surface water and cooling tower water specimens for Pseudomonas pseudomallei culture . The crude attack rate of melioidosis during the outbreak was 52 per 100,000 . Age, gender, race, diabetes and alcohol abuse were independent risk factors for disease . The relative risk of disease in diabetic patients was 12.9 (95% CI 5.1-32.7; p < 0.001) and 6.7 in alcoholic patients (95% CI 2.9-15.2; p < 0.001) . We found no significant difference between cases and controls in matched pair analysis for any of several exposure factors studied . We isolated Pseudomonas pseudomallei from 4% of soil samples and 9% of surface water samples . Our study confirms the importance of host factors in the development of melioidosis, and attempts to quantify the risk of disease during the Darwin epidemic . Pseudomonas pseudomallei is widespread in the soil of urban Darwin.

J Appl Bacteriol, 1993 Sep, 75(3), 292 - 8
Biological specificity of bottled natural mineral waters: characterization by ribosomal ribonucleic acid gene restriction patterns; Guillot E et al.; The organisms from heterotrophic plate counts of four brands of bottled non-carbonated mineral waters were analysed by ribosomal ribonucleic acid gene restriction patterns in addition to identification by customary techniques . Five commercialized bottles of each brand were examined once in the year 1989-90 . The total bacterial flora was divided into two main groups: the pseudomonads and the unidentified strains . Whereas phenotypic results did not reveal any significant differences between the four brands, rRNA gene restriction patterns were discriminatory . Among the 73 strains studied, 45 distinct patterns were observed, with only three common to several springs . These results reveal, at one determined time, a remarkable biological specificity of each brand of water.

Gene, 1993 Aug 16, 130(1), 57 - 63
Cloning and sequence analysis of a hemolysin-encoding gene from Pseudomonas paucimobilis; Minnick MF et al.; We report the cloning, expression and nucleotide (nt) sequence of a beta-hemolysin-encoding gene, termed hlyA, from Pseudomonas paucimobilis . A genomic DNA library of the pseudomonad was constructed in Escherichia coli using the plasmid vector, pUC19 . The hlyA gene was cloned by screening for a beta-hemolytic phenotype in E . coli transformants and was mapped to a 1100-bp PstI-SmaI fragment . The nt sequence analysis of the 1100-bp insert revealed a 789-bp open reading frame which is preceded by a 10-nt purine-rich sequence with a possible ribosome-binding site of GGA . The ORF terminates with a single UGA stop codon and is immediately followed by a large inverted repeat with 27-bp arms which may serve as a Rho-factor-independent transcriptional terminator . The hlyA gene codes for a protein of 263 amino acids (aa) residues with a deduced relative molecular mass (M(r)) of 29,695 and a predicted pI value of 11.5 . Expression of hlyA from recombinant DNA in E . coli occurred regardless of insert orientation in the vector and produced a 29-kDa protein . Confirmation of P . paucimobilis as the source of the cloned hlyA gene was determined by DNA hybridization . A search of various nt and aa sequence databases revealed no homologues to hlyA or its encoded protein.

Gene, 1993 Aug 16, 130(1), 47 - 55
Genetic analysis of a Pseudomonas locus encoding a pathway for biphenyl/polychlorinated biphenyl degradation; Hofer B et al.; The cistronic organization of the bph locus, encoding a biphenyl/polychlorinated biphenyl (PCB) degradation pathway in Pseudomonas sp . LB400, has been elucidated . Seven structural genes, encoding biphenyl dioxygenase (bphA1A2A3A4), biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (bphB), biphenyl-2,3-diol-1,2-dioxygenase (bphC) and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (bphD), have been located . The complete sequences of bphB, bphC and bphD are reported . Taken together with the data of Erickson and Mondello {J . Bacteriol . 174 (1992) 2903-2912}, Pseudomonas sp . LB400 is now the first strain for which the sequences of all genes encoding the catabolism from biphenyls to benzoates have been determined . Comparisons of the deduced amino acid (aa) sequences of BphB, BphC and BphD with those of related proteins led to predictions about catalytically important aa residues . Six Bph have been detected and identified . Five of them could be obtained as the most abundant proteins when their genes were expressed in Escherichia coli.

Gene, 1993 Aug 16, 130(1), 137 - 40
Cloning and expression of a Pseudomonas 3 alpha-hydroxysteroid dehydrogenase-encoding gene in Escherichia coli; Suzuki K et al.; A bacterial strain, B-0831, which produced 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD) was isolated and identified as belonging to the genus, Pseudomonas . Molecular weights of the purified 3 alpha HSD, determined by SDS-PAGE and by chromatography on Sephacryl S-200, were about 25 and 50 kDa, respectively . A genomic library of Pseudomonas sp . B-0831, prepared in the plasmid vector pACYC184, was screened with probes based on the amino acid (aa) sequence of the protein to obtain the plasmid, p3 alpha HSD1, identified by hybridization with the probes, that contained a 2.4-kb insert from Pseudomonas DNA . When the 1.4-kb SphI fragment of p3 alpha HSD1 was inserted into the vector, pUC118, and introduced into Escherichia coli DH1 under the control of lacZ promoter in the vector, the transformants produced 200-fold more 3 alpha HSD intracellularly than Pseudomonas sp . B-0831 . Sequence analysis of the 3 alpha HSD gene revealed that an ORF encoding 3 alpha HSD consists of 254 aa, with a calculated M(r) of 25,761, suggesting that the enzyme consists of homodimer subunits.

Gene, 1993 Aug 16, 130(1), 131 - 5
Chloroperoxidase-encoding gene from Pseudomonas pyrrocinia: sequence, expression in heterologous hosts, and purification of the enzyme; Wolfframm C et al.; The nucleotide sequence of a 1.5-kb fragment of Pseudomonas pyrrocinia DNA containing the chloroperoxidase(CPO)-encoding gene (cpo) and its flanking regions was determined . The cpo codes for a protein of 278 amino acids (aa) . The mature enzyme contains no N-terminal methionine, so that the CPO monomer consists of 277 aa with a calculated M(r) of 30,304 . Expression studies showed that the cpo from P . pyrrocinia is functionally expressed in Escherichia coli and Streptomyces lividans . Based on the overproduction of the CPO in E . coli, a novel and simple purification procedure was developed allowing the isolation of about 800-fold more CPO per gram of cells than was originally isolated from P . pyrrocinia . Comparison with the aa sequence of the bromoperoxidase BPO-A2 from S . aureofaciens ATCC10762 revealed an identity of 38%.

Cancer Res, 1993 Aug 15, 53(16), 3784 - 8
Eradication of small cell lung cancer cells from human bone marrow with immunotoxins; Myklebust AT et al.; The potential of autologous bone marrow transplantation to improve the treatment results for patients with small cell lung cancer (SCLC) may be limited by the presence of tumor cells in the graft . We constructed immunotoxins (ITs) involving 4 monoclonal antibodies and Pseudomonas exotoxin A and investigated the cytotoxicity of the ITs to H-146 SCLC cells in the presence and absence of normal human bone marrow (BM) cells . The Pseudomonas exotoxin A conjugate with the MOC-1 antibody, which recognizes an NCAM antigen, was inactive, as tested in a reproducible soft agar assay . Conjugates involving the monoclonal antibodies MOC-31, NrLu10, and MLuC1 killed about 3.5 log tumor cells at 0.1 microgram/ml and > 5.0 log at 1 microgram/ml . In the absence of BM cells, the combination of the 3 ITs was not superior to each IT used individually . However, when H-146 cells were admixed to nucleated BM cells at the ratio of 1:10, > 5 log tumor cell kill was obtained at a concentration as low as 0.1 microgram/ml of each IT . Survival of normal BM progenitor cells was only moderately reduced by the IT treatment, even in experiments in which the 3 IT3s were used at 2.5 micrograms/ml each . Freezing and thawing of the BM, as required in a clinical setting, reduced the colony-forming unit, granulocyte-macrophage, and colony-forming unit, granulocyte-erythroid-macrophage-megakaryocyte, by 30-60% in both treated and untreated cultures . We conclude that the use of a mixture of the 3 ITs provides a safe, rapid, and effective method for eradicating SCLC cells from BM used for autologous bone marrow transplantation following high-dose chemotherapy.

Proc Natl Acad Sci U S A, 1993 Aug 15, 90(16), 7774 - 8
The N-terminal region of the 37-kDa translocated fragment of Pseudomonas exotoxin A aborts translocation by promoting its own export after microsomal membrane insertion; Theuer CP et al.; The 37-kDa C-terminal fragment of Pseudomonas exotoxin A (PE; termed PE37 and composed of aa 280-613 of PE) translocates to the cell cytosol to cause cell death . PE37 requires a C-terminal endoplasmic reticulum retention sequence to be cytotoxic, indicating that the toxin may translocate to the cytosol from the endoplasmic reticulum . We show here that the N-terminal region of nascent PE37 can be inserted into the membrane of canine pancreatic microsomes by the preprocecropin signal sequence but then is exported or released from microsomes . The 34 N-terminal amino acids of the toxin fragment are sufficient to arrest translocation and prevent the microsomal accumulation of nascent chains that otherwise are sequestered into microsomes . These data support a role for the N-terminal region of PE37 in the translocation of the toxin from the endoplasmic reticulum to the cytosol in mammalian cells.

Proc Natl Acad Sci U S A, 1993 Aug 15, 90(16), 7538 - 42
A recombinant immunotoxin containing a disulfide-stabilized Fv fragment; Brinkmann U et al.; B3(dsFv)-PE38KDEL is a recombinant immunotoxin composed of the Fv region of monoclonal antibody B3 connected to a truncated form of Pseudomonas exotoxin (PE38KDEL), in which the unstable Fv heterodimer (composed of heavy- and light-chain variable regions) is held together and stabilized by a disulfide bond {termed disulfide-stabilized Fv (dsFV)} . A computer modeled structure of the B3(Fv), made by mutating and energy minimizing the amino acid sequence and structure of McPC603, enabled us to identify positions in conserved framework regions that "hypothetically" could be used for disulfide stabilization without changing the structure or affecting antigen binding . This prediction was evaluated experimentally by constructing a disulfide-linked two-chain dsFv-immunotoxin that was produced in Escherichia coli . The activity and specificity of this immunotoxin was indistinguishable from its single-chain Fv (scFv) counterpart, indicating that, as in B3(scFv), the structure of the binding region is retained in B3(dsFv) . Because we introduced the stabilizing disulfide bond in between two framework residues in a position that is conserved in most Fv molecules, this method of linkage between the heavy- and light-chain variable regions should be generally applicable to construct immunotoxins and dsFv molecules using other antibodies . Furthermore, the finding that B3(dsFv) was much more stable at 37 degrees C in human plasma than B3(scFv) indicates that dsFvs are possibly more versatile for therapeutic application than scFvs.

Mol Microbiol, 1993 Aug, 9(3), 579 - 89
An accessory gene, lipB, required for the production of active Pseudomonas glumae lipase; Frenken LG et al.; Pseudomonas glumae PG1 is able to secrete lipase into the extracellular medium . The lipase is produced as a precursor protein, with an N-terminal signal sequence . A second open reading frame (ORF) was found immediately downstream of the lipase structural gene, lipA, a situation found for the lipases of some other Pseudomonas species . Inactivation of this ORF resulted in a lipase-negative phenotype, indicating its importance in the production of active extracellular lipase . The ORF, lipB, potentially encodes a protein of 353-amino-acid residues, having a hydrophobic N-terminal (amino acids 1 to 90) and a hydrophilic C-terminal part . As a first step in determining the role of LipB, its subcellular location was determined . The protein was found to fractionate with the inner membranes . The expression of fusions of lipB fragments with phoA revealed an N(in)-C(out) topology for the LipB protein, which was confirmed by protease accessibility studies on EDTA-permeabilized cells and on inverted inner membrane vesicles . These and other results indicate that most of the LipB polypeptide is located in the periplasm and anchored to the inner membrane by an N-terminal transmembrane helix, located between amino acids 19 and 40.

J Antibiot (Tokyo), 1993 Aug, 46(8), 1203 - 7
Mureidomycins E and F, minor components of mureidomycins; Isono F et al.; Mureidomycins (MRDs) E and F were isolated from a culture filtrate of Streptomyces flavidovirens SANK 60486 which produces MRDs A approximately D . They possessed the same molecular formulae, C39H48N8O12S and very similar UV, IR and NMR spectra, but differed clearly from each other in HPLC profile . From the hydrolysates of MRDs E and F, 8-hydroxy-1,2,3,4-tetrahydro-3-isoquinoline carboxylic acid and 6-hydroxy-1,2,3,4-tetrahydro-3-isoquinoline carboxylic acid were detected, respectively, which were not detected from those of MRDs A approximately D . They showed strong anti-pseudomonal activity but less active than MRD A . MRDs E and F were synthesized from MRD A and formaldehyde through Pictet-Spengler reaction.

Plant Cell, 1993 Aug, 5(8), 865 - 75
RPS2, an Arabidopsis disease resistance locus specifying recognition of Pseudomonas syringae strains expressing the avirulence gene avrRpt2; Kunkel BN et al.; A molecular genetic approach was used to identify and characterize plant genes that control bacterial disease resistance in Arabidopsis . A screen for mutants with altered resistance to the bacterial pathogen Pseudomonas syringae pv . tomato (Pst) expressing the avirulence gene avrRpt2 resulted in the isolation of four susceptible rps (resistance to P . syringae) mutants . The rps mutants lost resistance specifically to bacterial strains expressing avrRpt2 as they retained resistance to Pst strains expressing the avirulence genes avrB or avrRpm1 . Genetic analysis indicated that in each of the four rps mutants, susceptibility was due to a single mutation mapping to the same locus on chromosome 4 . Identification of a resistance locus with specificity for a single bacterial avirulence gene suggests that this locus, designated RPS2, controls specific recognition of bacteria expressing the avirulence gene avrRpt2 . Ecotype Wu-0, a naturally occurring line that is susceptible to Pst strains expressing avrRpt2, appears to lack a functional allele at RPS2, demonstrating that there is natural variation at the RPS2 locus among wild populations of Arabidopsis.

J Clin Microbiol, 1993 Aug, 31(8), 2236 - 7
Tentative interpretive criteria for disk diffusion susceptibility testing of sparfloxacin; Fuchs PC et al.; Susceptibility to sparfloxacin was tested simultaneously by the agar dilution and disk diffusion methods . For tests with 5-micrograms sparfloxacin disks, we propose that the breakpoints for the susceptibility and resistance categories should be > or = 19 and < or = 15 mm, respectively . Most minor discrepancies involved tests with pseudomonads.

Lipids, 1993 Aug, 28(8), 721 - 6
The role of arginines in stabilizing the active open-lid conformation of Rhizomucor miehei lipase; Holmquist M et al.; Molecular dynamics simulations for the lid covering the active site of Rhizomucor miehei lipase {EC 3.1.1.3} postulated that, among other interactions, Arg86 in the lid stabilized the open-lid conformation of the protein by multiple hydrogen bonding to the protein surface . Chemical modification of arginine residues in R . miehei lipase with 1,2-cyclohexanedione or phenylglyoxal resulted in residual activities in the hydrolysis of tributyrin of 66 and 46%, respectively . Tryptic maps of native and phenylglyoxal-reacted R . miehei lipase showed that Arg86 was the residue modified most, when the lipase was inhibited to the greatest extent . Guanidine, a structural analog to an arginine side chain, inhibited both the native enzyme and the arginine-modified enzymes, resulting in residual activities of 26% as compared to the native enzyme . The inhibition was not an effect of enzyme denaturation . The native enzyme was also inhibited by 1-ethylguanidine, benzamidine and urea, but to a lesser degree than by guanidine . Lipases from Humicola lanuginosa and porcine pancreas in 100 mM guanidine showed residual activities of 88 and 70%, respectively . The lipases from Candida antarctica, C . rugosa, Pseudomonas cepacia and P . fluorescens were not inhibited by guanidine . The inhibition of R . miehei lipase by structural analogs of the arginine side chain and after chemical modification of arginine residues suggest a role of an arginine residue in stabilizing the active open-lid conformation of the enzyme.

Appl Environ Microbiol, 1993 Aug, 59(8), 2520 - 5
Degradation of nitrobenzene by a Pseudomonas pseudoalcaligenes; Nishino SF et al.; A Pseudomonas pseudoalcaligenes able to use nitrobenzene as the sole source of carbon, nitrogen, and energy was isolated from soil and groundwater contaminated with nitrobenzene . The range of aromatic substrates able to support growth was limited to nitrobenzene, hydroxylaminobenzene, and 2-aminophenol . Washed suspensions of nitrobenzene-grown cells removed nitrobenzene from culture fluids with the concomitant release of ammonia . Nitrobenzene, nitrosobenzene, hydroxylaminobenzene, and 2-aminophenol stimulated oxygen uptake in resting cells and in extracts of nitrobenzene-grown cells . Under aerobic and anaerobic conditions, crude extracts converted nitrobenzene to 2-aminophenol with oxidation of 2 mol of NADPH . Ring cleavage, which required ferrous iron, produced a transient yellow product with a maximum A380 . In the presence of NAD, the product disappeared and NADH was produced . In the absence of NAD, the ring fission product was spontaneously converted to picolinic acid, which was not further metabolized . These results indicate that the catabolic pathway involves the reduction of nitrobenzene to nitrosobenzene and then to hydroxylaminobenzene; each of these steps requires 1 mol of NADPH . An enzyme-mediated Bamberger-like rearrangement converts hydroxylaminobenzene to 2-aminophenol, which then undergoes meta ring cleavage to 2-aminomuconic semialdehyde . The mechanism for release of ammonia and subsequent metabolism are under investigation.

J Bacteriol, 1993 Aug, 175(15), 4859 - 69
Molecular analysis of avirulence gene avrRpt2 and identification of a putative regulatory sequence common to all known Pseudomonas syringae avirulence genes; Innes RW et al.; The avrRpt2 locus from Pseudomonas syringae pv . tomato causes virulent strains of P . syringae to be avirulent on some, but not all, lines of Arabidopsis thaliana and Glycine max (soybean) . We determined the DNA sequence of the avrRpt2 locus and identified the avrRpt2 gene as a 768-bp open reading frame encoding a putative 28.2-kDa protein . Deletion analysis and transcription studies provided further evidence that this open reading frame encodes AvrRpt2 . We found that the avrRpt2 gene also has avirulence activity in P . syringae pathogens of Phaseolus vulgaris (common bean), suggesting that disease resistance genes specific to avrRpt2 are functionally conserved among diverse plant species . The predicted AvrRpt2 protein is hydrophilic and contains no obvious membrane-spanning domains or export signal sequences, and there was no significant similarity of AvrRpt2 to sequences in the GenBank, EMBL, or Swiss PIR data bases . A comparison of the avrRpt2 DNA sequence to nine other P . syringae avirulence genes revealed a highly conserved sequence, GGAACCNA-N14-CCACNNA, upstream of the translation initiation codon . This motif is located 6 to 8 nucleotides upstream of the transcription start site in all four P . syringae avirulence genes for which a transcription start site has been determined, suggesting a role as a binding site for a novel form of RNA polymerase . Regulation of avrRpt2 was similar to other P . syringae avirulence genes; expression was high in minimal medium and low in rich medium and depended on the hrpRS locus and an additional locus at the opposite end of the hrp cluster of P . syringae pv . tomato.

Cell Struct Funct, 1993 Aug, 18(4), 241 - 51
Brefeldin A protects ricin-induced cytotoxicity in human cancer KB cell line, but not in its resistant counterpart with altered Golgi structures; Okimoto T et al.; Brefeldin A (BFA), an isoprenoid fungal metabolite, dramatically disrupts intracellular protein transport and protein secretion . BFA protects cells from the cytotoxicity of a plant toxin, ricin or pseudomonas toxin, but not that of diphtheria toxin (Yoshida et al., 1991 . Expt . Cell Res., 192: 389-395.) . In this study, we examined whether BFA could differentially change the cytotoxicity of ricin between BFA-sensitive cells and BFA-resistant cells . As a BFA-resistant cell line, we used a resistant cell line, KB/BF2-2, derived from BFA-sensitive human cancer KB cells . BFA treatment caused the disappearance of typical Golgi cisternae and the concomitant appearance of dilated vesicles in the cytoplasm in KB cells . By contrast, KB/BF2-2 cells had already altered Golgi structures with poor development of cisternae and also many vesicles in the absence of BFA, and BFA treatment did not further induce the morphological changes . Although a plasma membrane-specific marker protein, alpha-adaptin, was localized similarly in KB/BF2-2 as KB, Golgi specific markers such as beta-cop and gamma-adaptin were distributed in the cytoplasmic small vesicles as well as Golgi compartments in KB/BF2-2 cells in the absence of BFA, and the mutant cells showed no apparent changes in the distribution even when exposed to BFA . Ricin inhibited protein synthesis in KB and KB/BF2-2 to similar levels while pretreatment of KB cells with BFA at 0.1 microgram/ml almost completely reversed the inhibitory effect of ricin . By contrast, the pre-exposure of KB/BF2-2 cells to 1.0 microgram/ml BFA only partially rescued the ricin-induced inhibition of protein synthesis . Exposure to BFA at 30 min before ricin addition or at 0 min with ricin rescued the protein synthesis inhibition, but no rescue occurred when BFA was added 30 min after ricin addition . BFA could not rescue the protein synthesis inhibition by another toxin, diphtheria toxin . Our results suggest that BFA-resistant mutation causes a specific change in the endocytic membrane traffic of ricin in human cells, and also that cytotoxicity of diphtheria toxin does not share a common pathway of the intracellular transport with that of ricin.

Indian J Exp Biol, 1993 Aug, 31(8), 682 - 6
Patterns of heavy metal resistance in marine Pseudomonas MR1; Rani DB et al.; Pseudomonas strain MR1 isolated from the coastal waters of Bay of Bengal was found to resist Hg, As, Cd, Cu, Zn and Pb . It efficiently detoxified both organic and inorganic mercuric compounds to non toxic metallic mercury by an inducible enzyme, mercuric reductase . Its resistance to arsenic might be due to energy-dependent arsenate efflux system . Cadmium was detected intracellularly and in the surrounding medium . The bacterium accumulated copper and lead intracellularly.

Protein Eng, 1993 Aug, 6(6), 637 - 42
Pseudomonas glumae lipase: increased proteolytic stability by protein engineering; Frenken LG et al.; The feasibility of stabilizing proteins towards proteolytic degradation was explored by engineering the primary proteolytic cleavage site(s) . This novel approach does not require information on the 3-D structure of the native enzyme . As a model system, the extracellular lipase of Pseudomonas glumae was chosen, which is sensitive towards degradation by subtilisin-type proteases . The primary proteolytic cleavage in the lipase appeared to be located between amino acids serine 153 and histidine 154 . Since subtilisins are known to show a preference towards amino acid residues surrounding the scissile bond, non-preferred amino acids were introduced in this area . Two concepts were tested: the introduction of arginine or glutamate residues (charge concept) and the introduction of proline residues (proline concept) . Although the mutant lipases produced according to either of these concepts were still cleaved in the same area, they showed a considerably increased stability towards proteolytic degradation.

Plant J, 1993 Aug, 4(2), 327 - 41
Arabidopsis mutants compromised for the control of cellular damage during pathogenesis and aging; Greenberg JT et al.; Mutants of Arabidopsis thaliana which exhibit accelerated cell death in response to pathogens were isolated and characterized to gain insight into how symptom severity and disease resistance are modulated . This paper describes mutants that fall into one of two complementation groups that were identified . A novel feature of these mutants is that they are unable to control the rate and extent of cell death after exposure to a variety of stimuli that induce senescence responses . Thus, accelerated cell death (acd1) mutants show rapid, spreading necrotic responses to both virulent and avirulent Pseudomonas syringae pv . maculicola or pv . tomato pathogens and to ethylene . In addition, they develop necrotic lesions as they age and are sensitive to mechanical stress in a developmentally controlled manner . The acd1 mutants are also susceptible to opportunistic pathogens and show decreased growth inhibition of a heterologous pathogen of bean . The signal for lesion formation is not necessarily due to pathogens or wounding since plants grown aseptically also develop necrotic lesions . The lesions formed under a variety of conditions resemble those produced during a pathogen-induced rapid cell death response (the hypersensitive response, HR) . Analysis of these acd1 mutants may help to explain the molecular basis of the HR and the relationship between this response and the normal process of senescence.

J Gen Microbiol, 1993 Aug, 139 ( Pt 8), 1927 - 37
Molecular characterization of field isolates of Pseudomonas syringae pv . glycinea differing in coronatine production; Ullrich M et al.; Coronatine-producing and non-producing strains of Pseudomonas syringae pv . glycinea have been examined . We found a connection between copper resistance and synthesis of coronatine . Published data implied that these properties may be encoded on different plasmids . Production of coronatine and copper resistance were also found to be correlated for pv . glycinea in 19 field-isolates from leaf spots of plants in a soybean field and in 28 strains of a bacterial culture collection . Genomic diversity within pv . glycinea was investigated by plasmid profiling, DNA hybridization studies and PCR analysis . All strains unable to produce coronatine (cor-) were sensitive to copper ions and showed no homology to DNA from plasmid pSAY1, which carries a gene cluster for steps in coronatine production . In addition, cor- strains could be distinguished from coronatine-producing strains by a single unique band when amplified by random primer PCR . Plasmid profiles of strains isolated from field-populations during 1983, 1985 and 1990 showed that coronatine-producing and non-producing strains were present . The plasmid patterns also varied in 28 strains examined from a culture collection . No correlation between plasmid patterns and race specificity was observed . Cosmid pSAY1 proved to be an effective probe for detection of the coronatine synthesis genes and also revealed polymorphisms in coronatine producing strains of pv . glycinea.

Enzyme Microb Technol, 1993 Aug, 15(8), 634 - 45
Pseudomonas lipases: biochemical properties and molecular cloning; Gilbert EJ; Lipases (glycerol ester hydrolases; EC 3.1.1.3) are important enzymes which, due to their ability to catalyze a number of reactions, are receiving considerable interest from both academia and industry . The bacterial genus Pseudomonas is a prolific producer of a number of extracellular enzymes including lipase . This review summarizes the biochemical properties and recent advances in the molecular genetic analysis of a wide variety of Pseudomonas lipases . In particular, a comparison is made between the amino acid sequences of the various lipases as well as their secondary gene products, which are thought to be essential for secretion of the enzyme.

Appl Environ Microbiol, 1993 Aug, 59(8), 2746 - 9
Degradation of trichloroethylene by Pseudomonas cepacia G4 and the constitutive mutant strain G4 5223 PR1 in aquifer microcosms; Krumme ML et al.; Pseudomonas cepacia G4 degrades trichloroethylene (TCE) via a degradation pathway for aromatic compounds which is induced by substrates such as phenol and tryptophan . P . cepacia G4 5223 PR1 (PR1) is a Tn5 insertion mutant which constitutively expresses the toluene ortho-monooxygenase responsible for TCE degradation . In groundwater microcosms, phenol-induced strain G4 and noninduced strain PR1 degraded TCE (20 and 50 microM) to nondetectable levels (< 0.1 microM) within 24 h at densities of 10(8) cells per ml; at lower densities, degradation of TCE was not observed after 48 h . In aquifer sediment microcosms, TCE was reduced from 60 to < 0.1 microM within 24 h at 5 x 10(8) PR1 organisms per g (wet weight) of sediment and from 60 to 26 microM over a period of 10 weeks at 5 x 10(7) PR1 organisms per g . Viable G4 and PR1 cells decreased from approximately 10(7) to 10(4) per g over the 10-week period.

Infect Immun, 1993 Aug, 61(8), 3157 - 63
Role of a 22-kilodalton pilin protein in binding of Pseudomonas cepacia to buccal epithelial cells; Sajjan US et al.; Previously we have shown that many isolates of Pseudomonas cepacia obtained from the sputum of patients with cystic fibrosis exhibit specific binding to purified mucins . The binding was mediated by a 22-kDa protein located on peritrichous pili of the bacteria . Nonpiliated bacteria did not bind to mucin . In the present study we found that both piliated and nonpiliated P . cepacia bind to buccal epithelial cells (BECs) obtained from health human volunteers . Scatchard plot analyses of binding data with the LIGAND computer program suggest the presence of at least two classes of cell receptors (A and B) for piliated P . cepacia (isolates PC 5 and PC 7) and a single class of receptors (A) for nonpiliated P . cepacia (isolates PC 45 and PC 61) . The affinity constants for receptor A varied from 1.7 x 10(-9) to 4.7 x 10(-8) ml/CFU . Receptor B had a lower affinity constant (2.5 x 10(-10) to 1.2 x 10(-9) ml/CFU) but a greater saturation capacity . Receptor B was similar in affinity to the mucin receptor for piliated P . cepacia (3.3 x 10(-10) to 1.3 x 10(-9) ml/CFU) . Purified mucin partially inhibited the binding of piliated bacteria to BECs by competing with BEC receptor site B . The purified 22-kDa pilin adhesin and an antiadhesin antibody also caused partial inhibition . One BEC receptor for piliated isolates of P . cepacia was identified as a 55-kDa protein as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of BEC homogenate supernatants, Western blotting (immunoblotting), and bacterial overlay assays . Preincubation of piliated bacteria with either mucin or the antiadhesin antibody abolished binding to the 55-kDa BEC receptor . In summary, our results indicate that piliated P . cepacia interacts with BECs by using at least two different adhesin-receptor systems . One adhesin (not examined) is common to piliated and nonpiliated P . cepacia, but the other system is the pilus-localized 22-kDa mucin-binding adhesin and its 55-kDa BEC receptor protein . Because it mediates adherence to both mucin and epithelial cells, the 22-kDa adhesin may be an important virulence determinant in cystic fibrosis lung infections in which mucins are abnormally adhesive on mucosal surfaces.

Biochim Biophys Acta, 1993 Jul 18, 1174(1), 79 - 82
Cloning and sequence analysis of an esterase gene from Pseudomonas sp . KWI-56; Shimada Y et al.; The gene encoding an esterase from Pseudomonas sp . KWI-56 was cloned and sequenced . The nucleotide sequence contained an open reading frame encoding a polypeptide comprising 262 amino acids, whose molecular weight agreed well with the value obtained by SDS-PAGE . Comparison of the amino acid sequence with those of the other homologous enzymes suggested that Ser-92 and His-24l might be included in the catalytic triad.

J Biol Chem, 1993 Jul 5, 268(19), 14065 - 70
A wide range of human cancers express interleukin 4 (IL4) receptors that can be targeted with chimeric toxin composed of IL4 and Pseudomonas exotoxin; Debinski W et al.; A chimeric toxin has been constructed by fusion of a gene encoding human interleukin 4 (hIL4) to a gene encoding a mutant form of Pseudomonas exotoxin A (PE) which cannot bind to its receptors (PE4E) . The chimeric gene was expressed in Escherichia coli where large amounts of the chimeric toxin, hIL4-PE4E, was produced . Purified hIL4-PE4E was very cytotoxic to cancer cell lines of both hematopoietic and solid tumor origin . In the HUT 102 T cell leukemia and Daudi B cell lymphoma cell lines, protein synthesis was inhibited by 50% (ID50) at a hIL4-PE4E concentration of 2 and 7 ng/ml (25 and 86 pM, respectively) . hIL4-PE4E was also very cytotoxic to cell lines derived from carcinomas of the colon, breast, stomach, liver, adrenals, and prostate, as well as melanoma and epidermoid carcinoma, indicating that hIL4 receptors are widely expressed on human malignancies . We also found that human phytohemagglutinin-activated peripheral blood lymphocytes were extremely sensitive to hIL4-PE4E with an ID50 of 0.2 ng/ml (2.5 pM) . The cytotoxic action of hIL4-PE4E was specific because it was blocked by an excess of hIL4 and not of human interleukin 2 . In addition, hIL4-PE4ED553, an enzymatically inactive form of the chimeric toxin, was not cytotoxic . These results suggest that the hIL4 receptor may be a target for therapy in malignant and immunologic disorders using hIL4 chimeric toxin.

Lancet, 1993 Jul 3, 342(8862), 15 - 9
Evidence for transmission of Pseudomonas cepacia by social contact in cystic fibrosis; Govan JR et al.; Pulmonary colonisation with Pseudomonas cepacia in patients with cystic fibrosis can be associated with increased morbidity and mortality . The modes of transmission of P cepacia are, however, unclear . We used selective media and phenotypic and genomic typing systems to investigate the acquisition of P cepacia by adults with cystic fibrosis . An analysis of isolates from 210 patients attending regional clinics in Edinburgh and Manchester between 1986 and 1992 showed that the main cause of increased isolations of P cepacia from 1989 was the emergence of an epidemic strain that had spread between patients in both clinics . Epidemiological evidence indicated that social contact was important in spread of the epidemic strain within and between clinics . We suggest that guidelines to limit the acquisition of P cepacia should not be restricted to patients in hospital, and that intimate or frequent social contact is associated with a high risk of cross-infection.

Cell, 1993 Jul 2, 73(7), 1255 - 66
Pseudomonas syringae pv . syringae harpinPss: a protein that is secreted via the Hrp pathway and elicits the hypersensitive response in plants; He SY et al.; The ability of P . syringae to elicit the hypersensitive response in nonhost plants or pathogenesis in hosts is controlled by hrp genes . The P . syringae pv . syringae 61 hrpZ gene encodes harpinPss, a 34.7 kd extracellular protein that elicits hypersensitive necrosis in tobacco and other plants . HarpinPss is heat stable, glycine rich, dissimilar in amino acid sequence to any known protein, produced only in apoplastic fluid-mimicking minimal media, and secreted in a HrpH-dependent manner . The carboxy-terminal 148 amino acid portion of harpinPss contains two directly repeated sequences of GGGLGTP and QTGT and is sufficient and necessary for elicitor activity . The necrosis elicited by harpinPss is an active response of the plant, which can be inhibited by alpha-amanitin, cycloheximide, lanthanum chloride, or sodium vanadate.

Mol Plant Microbe Interact, 1993 Jul-Aug, 6(4), 434 - 43
Arabidopsis mutations at the RPS2 locus result in loss of resistance to Pseudomonas syringae strains expressing the avirulence gene avrRpt2; Yu GL et al.; We isolated and characterized two Arabidopsis thaliana mutants that fail to mount a hypersensitive defense response (HR) when infiltrated with phytopathogenic Pseudomonas strains carrying the avirulence (avr) gene avrRpt2 but still mount an HR when infiltrated with strains carrying other avr genes . One of these mutants was isolated using a method we developed that enriches for Arabidopsis seedlings that survive vacuum-infiltration with a bacterial strain carrying an avr gene . Genetic analysis showed that the phenotypes of both mutants resulted from mutations at a single locus, RPS2 . In contrast to the wild type, both rps2 mutants failed to limit the growth of Pseudomonas strains carrying avrRpt2 . Heterozygous RPS2/rps2 plants displayed a phenotype intermediate between those of RPS2/RPS2 and rps2/rps2 homozygotes . These experiments show that the wild-type allele at the rps2 locus, RPS2, encodes a component of a signal transduction pathway that responds to a signal generated by avrRpt2 and that RPS2 is required for the elicitation of an HR . RPS2 was mapped near the restriction fragment length polymorphism marker PG11 on chromosome IV.

Mol Gen Mikrobiol Virusol, 1993 Jul-Aug, (4), 37 - 40
{Transduction of Pseudomonas mallei bacteria}; Manzeniuk OIu et al.; The transducing ability of 17 Pseudomonas pseudomallei bacteriophages has been studied . Five of them were found to be capable of transducing the transposon Tn7 markers TpR, SmR into the strain Pseudomonas mallei C-5 . The transduced bacterial recipients became lysogenic and acquired resistance to trimethoprim and streptomycin . Transduction of transposon Tn7 has been confirmed by DNA-DNA hybridization.

J Gen Microbiol, 1993 Jul, 139 ( Pt 7), 1587 - 94
Differentiation of Pseudomonas solanacearum, Pseudomonas syzygii, Pseudomonas pickettii and the Blood Disease Bacterium by partial 16S rRNA sequencing: construction of oligonucleotide primers for sensitive detection by polymerase chain reaction; Seal SE et al.; The sequence of a 292 bp segment of the DNA encoding 16S rRNA (corresponding to positions 44-337 of the Escherichia coli 16S rRNA sequence) was determined for each of 40 Pseudomonas solanacearum, four banana Blood Disease Bacterium, three P . syzygii and two P . pickettii strains . Phylogenetic relationships derived from comparison of these sequences to each other, and to equivalent 16S rRNA gene sequences from other bacteria present in the EMBL databank, conform well with those obtained previously by DNA-DNA/rRNA hybridization experiments . The 16S rRNA sequence of the Blood Disease Bacterium was identical over the 292 bp to one of the four sequence groups of P . solanacearum, suggesting that these pseudomonads are more closely related to each other than to P . syzygii or P . pickettii . Sequence data comparisons allowed construction of an oligonucleotide specific for P . solanacearum, P . syzygii and the Blood Disease Bacterium . Use of the specific oligonucleotide with a non-specific oligonucleotide in the polymerase chain reaction enabled 1-10 cells of bacteria in this group to be detected after 50 rounds of amplification by visualizing a 287-288 bp product on agarose gels.

Can J Microbiol, 1993 Jul, 39(7), 659 - 64
The 73-kb pIAA plasmid increases competitive fitness of Pseudomonas syringae subspecies savastanoi in oleander; Silverstone SE et al.; Pseudomonas syringae subsp . savastanoi causes tumors on olive and oleander by producing the plant growth regulators indoleacetic acid (IAA) and cytokinins following infection of the plant . The contribution of IAA production to the ability of P . syringae subsp . savastanoi to grow and survive in oleander leaf tissue was studied . Bacterial strains differing only with respect to IAA production were characterized . Growth and survival of wild-type and two mutant strains of P . syringae subsp . savastanoi in oleander leaf tissue were monitored by weekly colony counts and IAA plate assays . Growth rate of the three strains in culture and in planta did not differ significantly . However, the wild-type strain reached a higher population density and maintained its maximum density at least 9 weeks longer than either mutant population . An insertion mutant containing the IAA plasmid (pIAA), but incapable of IAA production, did not maintain a higher population density than a strain cured of the IAA plasmid . The pIAA-cured strain maintained a higher population density when coinoculated with an IAA-producing strain than when inoculated alone . These results suggest that IAA production may contribute to the fitness of P . syringae subsp . savastanoi in oleander tissue and that the iaa operon alone may be responsible for the competitive advantage of cells harboring pIAA.

J Bacteriol, 1993 Jul, 175(14), 4561 - 4
Oxidation of polychlorinated biphenyls by Pseudomonas sp . strain LB400 and Pseudomonas pseudoalcaligenes KF707; Gibson DT et al.; Biphenyl-grown cells and cell extracts prepared from biphenyl-grown cells of Pseudomonas sp . strain LB400 oxidize a much wider range of chlorinated biphenyls than do analogous preparations from Pseudomonas pseudoalcaligenes KF707 . These results are attributed to differences in the substrate specificity of the biphenyl 2,3-dioxygenases from both organisms.

J Bacteriol, 1993 Jul, 175(14), 4492 - 8
Characterization of chromosomal homologs of the plasmid-borne copper resistance operon of Pseudomonas syringae; Lim CK et al.; Copper-resistant and copper-sensitive strains of Pseudomonas syringae, as well as many other pseudomonads, contain chromosomal DNA homologous to the plasmid-borne copper resistance operon (copABCD) . cop homologs were cloned from the chromosome of P . syringae pv . tomato PT12.2, which had an elevated level of resistance to copper compared with typical copper-sensitive strains of other P . syringae pathovars and showed an unusually high frequency of spontaneous mutation to high levels of copper resistance . Two chromosomal cop homolog regions were cloned . Homolog 1 hybridized with copA and copB, and homolog 2 hybridized with copA, copB, copC, and the copper-responsive regulatory genes copRS . Homolog 1 had no detectable function when transferred to a copper-sensitive strain of P . syringae . However, homolog 2 conferred the low level of copper resistance observed with PT12.2 and produced proteins related to CopA and CopC . In addition, homolog 2 conferred a high frequency of mutation to full copper resistance . In a spontaneously mutated derivative of the cloned homolog 2 (pCOPH2R) that conferred copper resistance, an increased level of CopA was observed . pCOPH2R also supported a higher level of transcriptional activity of the cop promoter that was fused to lacZ and provided in trans (pCOP38), suggesting that the spontaneous mutation was regulatory, probably involving the copRS homologs . Homolog 2 was similar but not identical to the plasmid-borne cop operon, and it did not complement site-specific mutations in cop genes.

Biochim Biophys Acta, 1993 Jul 1, 1168(3), 253 - 60
Determination of the lipase stereoselectivities using circular dichroism (CD); lipases produce chiral di-O-acylglycerols from achiral tri-O-acylglycerols; Uzawa H et al.; A general method was described to determine the optical purity of 1,2 (or 2,3)-di-O-acylglycerols via a key compound, 3 (or dibenzoyl-sn-glycerol (3 or 3') . The chiral di-O-acylglycerols were first silylated and the acyl groups were removed by the Grignard degradation to 3 (or 1) O-tert-butyldimethylsilyl-sn-glycerol and subsequent benzoylation lead to the key compound 3 or 3' without racemization . The optical purity was determined from the strong exciton Cotton effect of 3 (+) or 3' (-) at 238 nm in the concentration of ca . 1 mM . The method was successfully applied to determine the stereoselectivities of lipases (EC 3.1.1.3) from three origins, bacteria, mammal and fungus such as Pseudomonas (AP, 89% optical purity, sn-1 preference), porcine pancreatin (PPL, 9.3% optical purity, sn-3 preference) and Candida (CC, sn-2 preference) using tripalmitin . The similar studies were extended to tri-O-benzoylglycerol (6) and tri-O-(cyclohexanecarbonyl)glycerol (5) . All the enzymes showed high stereoselectivities with tri-O-benzoylglycerol . PPL and AP showed high and low stereoselectivities with tri-O-(cyclohexanecarbonyl)glycerol, while low and high stereoselectivities with tri-O-palmitoylglycerol, respectively . The results show that the stereoselectivities are ruled by the origins of lipases and acyl groups . The structures of the recognition site might be associated with enantioselectivities of the enzymes.

J Bacteriol, 1993 Jul, 175(13), 4062 - 70
High-level expression of ice nuclei in a Pseudomonas syringae strain is induced by nutrient limitation and low temperature; Nemecek-Marshall M et al.; Attempts were made to maximize the expression of ice nuclei in Pseudomonas syringae T1 isolated from a tomato leaf . Nutritional starvation for nitrogen, phosphorous, sulfur, or iron but not carbon at 32 degrees C, coupled to a shift to 14 to 18 degrees C, led to the rapid induction of type 1 ice nuclei (i.e., ice nuclei active at temperatures warmer than -5 degrees C) . Induction was most pronounced in stationary-phase cells that were grown with sorbitol as the carbon source and cooled rapidly, and under optimal conditions, the expression of type 1 ice nuclei increased from < 1 per 10(7) cells (i.e., not detectable) to 1 in every cell in 2 to 3 h . The induction was blocked by protein and RNA synthesis inhibitors, indicative of new gene expression . Pulse-labeling of nongrowing cultures with {35S}methionine after a shift to a low temperature demonstrated that the synthesis of a new set of "low-temperature" proteins was induced . Induced ice nuclei were stable at a low temperature, with no loss in activity at 4 degrees C after 8 days, but after a shift back to 32 degrees C, type 1 ice nuclei completely disappeared, with a half-life of approximately 1 h . Repeated cycles of low-temperature induction and high-temperature turnover of these ice nuclei could be demonstrated with the same nongrowing cells . Not all P . syringae strains from tomato or other plants were fully induced under the same culture conditions as strain T1, but all showed increased expression of type 1 ice nuclei after the shift to the low temperature . In support of this view, analysis of the published DNA sequence preceding the translational start site of the inaZ gene (R . L . Green and G . Warren, Nature {London} 317:645-648, 1985) suggests the presence of a gearbox-type promoter (M . Vincente, S . R . Kushner, T . Garrido, and M . Aldea, Mol . Microbiol . 5:2085-2091, 1991).

ASAIO J, 1993 Jul-Sep, 39(3), M668 - 74
Quantitative bacterial analysis of porous, fabric, and smooth non-blood contacting implant surfaces and their tissue interfaces in a 169 day pneumatic total artificial heart animal recipient; Seare WJ Jr et al.; All long-term total artificial heart (TAH) survivals are subject to sepsis . Survival can be prolonged, but the source of the infection cannot be eliminated with any known course of antibiotics or treatment regimen . Sambo, a U-100 pTAH calf, survived 169 days . At week 6, he became septic, growing a Pseudomonas species (Ps) . Weekly blood cultures were intermittently positive until week 13 when they became continuously positive until his demise, from a ruptured left ventricular pumping diaphragm . Spatially specific porous silicone rubber (SSP) was used for surface modifications on the drive lines and as cuffs around the Dacron TAH graft to large vessel anastomoses . This gave an excellent opportunity to examine two types of porous implants surfaces (Dacron grafts and SSP) to the smooth Biomer ventricular surfaces with their respective adjoining tissue interfaces for bacterial colonization . Nine tissue samples and 13 implant surfaces were processed with Costerton's quantitative bacterial techniques . The largest numbers of bacteria (> 10(6)/cm2 Ps.) were grown from the smooth ventricular surface and in the cul-de-sac where the SSP delaminated from the driveline (two smooth implant surfaces in contact but without tissue apposition) . The Dacron grafts were intermediate in bacterial concentrations and SSP surface modified drivelines and tissues were sterile . In this model, the more intimate biointegration found in the porous implants showed improved bacterial resistance in a chronically infected pTAH . The more completely biointegrated and neo-vascularized porosity SSP was the only implant surface and opposing implant tissue interface sampled to remain sterile.

Biol Chem Hoppe Seyler, 1993 Jul, 374(7), 489 - 96
Site-directed mutagenesis of the 2-haloalkanoic acid dehalogenase I gene from Pseudomonas sp . strain CBS3 and its effect on catalytic activity; Schneider B et al.; Two 2-haloalkanoic acid dehalogenases from Pseudomonas sp . strain CBS3 catalyse hydrolytic dehalogenation of chloroacetate and 2-chloropropionate . We used site-directed mutagenesis to introduce specific changes in the dehalogenase I encoding gene (dehCI) . Substitution of Asp-10 by Ala-10 resulted in complete loss of dehalogenating activity although expression of the 2-haloacid dehalogenase I was not affected in the mutant as shown by western blot analysis, and although comparison of the mutated enzyme with the wild type enzyme indicated that extensive rearrangements in the three-dimensional structure of the enzyme had not occurred . From these data we suggest that Asp-10 of 2-haloacid dehalogenases I from Pseudomonas sp . strain CBS3 may be the nucleophilic residue in the active-site of this enzyme essential for halide release.

Kidney Int, 1993 Jul, 44(1), 191 - 8
Outcome of HIV infected patients on continuous ambulatory peritoneal dialysis; Tebben JA et al.; A retrospective analysis of 39 HIV infected patients with ESRD cared for in New Haven from 1987 to June 1992 was performed . All patients had evidence for HIV infection at the start of CAPD therapy . Cumulative technique survival at one and two years was 43% and 27%, respectively . Only eight patients transferred to center dialysis . One and two year patient survival on CAPD was 58% and 54%, respectively . Mortality was higher in patients with advanced infection than in those with asymptomatic HIV infection . Hospitalization rates were also higher in patients with advanced infection . HIV infected patients had higher rates of peritonitis (3.9 episodes/outpatient CAPD year) compared to non-HIV infected patients (1.5 episodes/CAPD year), especially for pseudomonal and fungal infections . Active injection drug use and use of the "straight set" system were associated with increased rates of peritonitis . CAPD deserves consideration as a therapy for HIV infected patients with ESRD.

Lett Appl Microbiol, 1993 Jul, 17(1), 44 - 8
Restriction analysis of an amplified polygalacturonase gene fragment differentiates strains of the phytopathogenic bacterium Pseudomonas solanacearum; Gillings M et al.; Amplification of a polygalacturonase gene fragment using the polymerase chain reaction (PCR) formed a rapid, sensitive and portable method for detecting and differentiating strains of Pseudomonas solanacearum, a taxonomically complex bacterial species . Primers 5'CAG CAG AAC CCG CGC CTG ATC CAG 3' and 5'ATC GGA CTT GAT GCG CAG GCC GTT 3' were used to amplify a 504 base pair polygalacturonase gene fragment from 57 Ps . solanacearum isolates . Digestion of these products with Hae III defined groups which reflected the known genetic divisions within the species.

Plant Physiol, 1993 Jul, 102(3), 891 - 901
Complementary immunolocalization patterns of cell wall hydroxyproline-rich glycoproteins studied with the use of antibodies directed against different carbohydrate epitopes; Swords KM et al.; Antisera raised against the major hydroxyproline-rich glycoprotein (HRGP) in carrot (Daucus carota L.) taproot, extensin-1, and a minor HRGP, extensin-2, were characterized by western blot analysis, enzyme-linked immunosorbent assay, and periodate oxidation and found to be directed against carbohydrate epitopes shared by both glycoproteins . The anti-extensin-1 antibodies (gE1) target periodate-sensitive epitopes and may recognize the terminal alpha-1,3-arabinoside of extensin-1 . The anti-extensin-2 antibodies (gE2) recognize periodate-insensitive epitopes, possibly binding the reducing, internal beta-1,2-arabinosides on the carbohydrate side chains . Despite the cross-reactivity of these antibodies, immunolocalization studies of carrot taproot and green bean (Phaseolus vulgaris L.) leaf tissues reveal a spatial segregation of gE1- and gE2-labeling patterns . The gE1 antibodies bind only to the cellulose-rich region of the cell wall (J.P . Staehelin and L.A . Stafstrom {1988} Planta 174: 321-332), whereas gE2 labeling is restricted to the expanded middle lamella at three cell junctions . Periodate oxidation of nonosmicated, thin-sectioned tissue abolishes gE1 labeling but leads to labeling of the entire cell wall by gE2, presumably as a result of unmasking cryptic epitopes on extensin-1 in the cellulose layer . Purified extensin-2 protein is more efficient than extensin-1 protein at agglutinating avirulent Pseudomonas strains lacking extracellular polysaccharide . Our results indicate that extensin-2 does not form a heterologous HRGP network with extensin-1 and that, in contrast to extensin-1, which appears to serve a structural role, extensin-2 could participate in passive defense responses against phytopathogenic bacteria.

Ann N Y Acad Sci, 1993 Jun 23, 685, 740 - 5
Pseudomonas exotoxin and recombinant immunotoxins derived from it; Fitzgerald D et al.; Pseudomonas exotoxin (PE) is a bacterial toxin that kills mammalian cells by gaining entry to the cytosol and inactivating protein synthesis . The toxin binds and enters cells via the alpha 2-macroglobulin receptors . Within cells, the toxin is processed in several steps to produce an enzymatically active 37-kDa C-terminal fragment which translocates to the cytosol and ADP-ribosylates elongation factor 2 . Because PE is a very potent toxin, derivatives of it have been produced which, when joined to various binding ligands, are capable of killing specific target cells . It is hoped that this strategy will lead to the development of effective therapeutic agents for the treatment of human diseases such as cancer, AIDS, and various immunologic disorders.

J Biol Chem, 1993 Jun 15, 268(17), 12596 - 602
Cerulenin inhibits the cytotoxicity of ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in brefeldin A-resistant cell lines; Oda T et al.; We have found that cerulenin, an antibiotic that inhibits de novo fatty acid and cholesterol biosynthesis and fatty acylation of proteins, strongly inhibited the cytotoxicity of ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in a brefeldin A (BFA)-resistant mutant of Vero cells (BER-40) . The protective effect of cerulenin against ricin was also observed in two other BFA-resistant cell lines, Madin-Darby canine kidney, and PtK1 cells . In contrast to BER-40 cells, no significant effect of cerulenin was observed in Vero cells . Cerulenin did not affect the binding of ricin to the cell-surface receptors, but reduced significantly the internalization of ricin in BER-40 cells; no effect of cerulenin on the binding or internalization of ricin was observed in Vero, PtK1, and Madin-Darby canine kidney cells . Endocytic uptake of fluid-phase markers such as horseradish peroxidase and lucifer yellow was inhibited by cerulenin in BER-40 cells, but the endocytosis of transferrin via the coated pit/coated vesicle pathway was slightly increased . Cerulenin inhibited the degradation and excretion of ricin in BER-40 cells, and this effect of cerulenin was not observed in Vero cells . Furthermore, cerulenin inhibited the bulk protein secretion in a dose-dependent manner, with BER-40 cells being more susceptible than Vero cells . These results suggest that in addition to its effect on endocytosis, cerulenin interferes with the intracellular trafficking or processing of toxin molecules, and the vesicle transport system in BER-40 cells appears to be cerulenin-sensitive . Since addition of fatty acids and cholesterol did not reverse the effects of cerulenin, the protective effect of cerulenin against protein toxins is not due to an inhibition of de novo fatty acids and cholesterol biosynthesis.

Gene, 1993 Jun 15, 128(1), 5 - 11
Structural constraints on the display of foreign peptides on filamentous bacteriophages; Makowski L; Strategies for the construction of vehicles for phage display are evaluated here on the basis of structural studies of filamentous bacteriophages . Potential sites for the insertion of foreign peptides into the major coat protein, gp8, of M13 are identified . Currently, the insertion of peptides into gp8 has two basic limitations: all insertion sites that have been used successfully are located within 5 amino acids (aa) of the N terminus, and in virions containing only mutant coat proteins, insertions larger than about 6 aa have not been successfully incorporated . The possible reasons for these limitations are discussed in terms of the structures of gp8 and the minor structural proteins, gp7 and gp9 . Potential strategies for overcoming these limitations are outlined . Reasons for the successful incorporation of larger inserts into hybrid phage containing both native and mutant coat proteins are also discussed . The structures of gp6, gp7, and gp9 are described, and it is concluded that insertion sites in these minor proteins are unlikely to have substantial advantages over those currently being used in gp3 . The structure of the coat protein of another filamentous phage, Pseudomonas phage Pf1, is also described . Its structure provides a number of clues for the successful design of phage display insertion sites . Because it contains a 7-aa surface loop in the major coat protein, the Pf1 coat protein may have significant advantages over gp8 of M13 as a vehicle for phage display.

Biochem J, 1993 Jun 15, 292 ( Pt 3), 697 - 700
A class-A beta-lactamase from Pseudomonas stutzeri that is highly active against monobactams and cefotaxime; Franceschini N et al.; A beta-lactamase produced by Pseudomonas stutzeri was purified to protein homogeneity, and its physicochemical and catalytic properties were determined . Its profile was unusual since, in addition to penicillins, the enzyme hydrolysed second- and third-generation 'beta-lactamase-stable' cephalosporins and monobactams with similar efficiencies . On the basis of the characteristics of the interaction with beta-iodopenicillanic acid, the enzyme could be classified as a class-A beta-lactamase . However, when compared with most class-A beta-lactamases, it exhibited significantly lower kcat./Km values for the compounds usually considered to be the best substrates of these enzymes.

Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5682 - 6
Activation of a bacterial lipase by its chaperone; Hobson AH et al.; The gene lipA of Pseudomonas cepacia DSM 3959 encodes a prelipase from which a signal peptide is cleaved during secretion, producing a mature extracellular lipase . Expression of lipase in several heterologous hosts depends on the presence of another gene, limA, in cis or in trans . Lipase protein has been overproduced in Escherichia coli in the presence and absence of the lipase modulator gene limA . Therefore, limA is not required for the transcription of lipA or for the translation of the lipA mRNA . However, no lipase activity is observed in the absence of limA . limA has been overexpressed and encodes a 33-kDa protein, Lim . If lipase protein is denatured in 8 M urea and the urea is removed by dialysis, lipase activity is quantitatively recovered provided Lim protein is present during renaturation . Lip and Lim proteins form a complex precipitable either by an anti-lipase or anti-Lim antibody . The Lim protein has therefore the properties of a chaperone.

Biochim Biophys Acta, 1993 Jun 11, 1157(2), 119 - 26
Role of Hageman factor/kallikrein-kinin system in pseudomonal elastase-induced shock model; Khan MM et al.; The role of the Hageman factor dependent pathway in pseudomonal elastase-induced shock was investigated in guinea pigs . Presence of a bradykinin B2 receptor antagonist {D-Arg0,Hyp3,Thi5,8,D-Phe7}-bradykinin (200 nM) in the circulation prevented shock caused by an intrajugular injection of pseudomonal elastase (0.8 mg/kg body weight) . During the lethal shock caused by elastase (1.2 mg/kg), a significant consumption of components of the Hageman factor/kallikrein-kinin system was observed such as 45.7 +/- 2.20% consumption of Hageman factor, 100 +/- 0% of prekallikrein, and 85.1 +/- 2.50 of high-molecular-weight kininogen . More striking evidence for the participation of this system was demonstrated in depletion experiments with monospecific F(ab')2 antibodies against the components of the system . After depletion of any one of the components, guinea pigs exhibited unresponsiveness to the same lethal dose of pseudomonal elastase in regard to the cardio-respiratory alterations . In vitro, pseudomonal elastase (60 micrograms/ml) possessed a capacity to generate substantial amount of bradykinin in undiluted plasmas of humans (300.0 +/- 32.16 ng/ml) as well as guinea pigs (460.2 +/- 20.67 ng/ml) at 37 degrees C but not in those deficient in Hageman factor or prekallikrein . These results strongly suggested a pathological role of elastase in pseudomonal sepsis through activation of the Hageman factor dependent pathway.

J Bacteriol, 1993 Jun, 175(12), 3703 - 9
Developmental regulation of the gene for formate dehydrogenase in Neurospora crassa; Chow CM et al.; We have isolated and characterized a gene, fdh, from Neurospora crassa which is developmentally regulated and which produces formate dehydrogenase activity when expressed in Escherichia coli . The gene is closely linked (less than 0.6 kb apart) to the leu-5 gene encoding mitochondrial leucyl-tRNA synthetase; the two genes are transcribed convergently from opposite strands . The expression patterns of these genes differ: fdh mRNA is found only during conidiation and early germination and is not detectable during mycelial growth, while leu-5 mRNA appears during germination and mycelial growth . The structure of the fdh gene was determined from the sequence of cDNA and genomic DNA clones and from mRNA mapping studies . The gene encodes a 375-amino-acid-long protein with sequence similarity to NAD-dependent dehydrogenases of the E . coli 3-phosphoglycerate dehydrogenase (serA gene product) subfamily . In particular, there is striking sequence similarity (52% identity) to formate dehydrogenase from Pseudomonas sp . strain 101 . All of the residues thought to interact with NAD in the crystal structure of the Pseudomonas enzyme are conserved in the N . crassa enzyme . We have further shown that expression of the N . crassa gene in E . coli leads to the production of formate dehydrogenase activity, indicating that the N . crassa gene specifies a functional polypeptide.

J Urol, 1993 Jun, 149(6), 1626 - 32
A recombinant form of Pseudomonas exotoxin A containing transforming growth factor alpha near its carboxyl terminus for the treatment of bladder cancer; Theuer CP et al.; The epidermal growth factor receptor (EGFR) is overexpressed on the superficial layers of malignant urothelium and is suspected of playing a role in tumor progression . TP40 is a chimeric protein composed of transforming growth factor-alpha (TGF alpha) fused to a modified form of Pseudomonas exotoxin A (PE) that is selectively cytotoxic to EGFR-bearing cells and is currently undergoing clinical study for the intravesical therapy of bladder cancer . We constructed a recombinant toxin PE35/TGF alpha-KDEL as an improved agent for the local therapy of EGFR-bearing bladder cancer . PE35/TGF alpha-KDEL does not require intracellular proteolysis to generate a carboxyl-terminal fragment capable of reaching the target cell cytosol and contains a modified carboxyl-terminal sequence KDEL, that increases toxin activity . These features make PE35/TGF alpha-KDEL from 10- to 700-fold more potent than TP40 on four human bladder cancer cell lines . PE35/TGF alpha-KDEL may be a useful agent for treatment of EGFR-bearing cancers.

Mol Microbiol, 1993 Jun, 8(6), 1039 - 51
The alkane oxidation system of Pseudomonas oleovorans: induction of the alk genes in Escherichia coli W3110 (pGEc47) affects membrane biogenesis and results in overexpression of alkane hydroxylase in a distinct cytoplasmic membrane subfraction; Nieboer M et al.; The alkane hydroxylase system of Pseudomonas oleovorans, which catalyses the initial oxidation of aliphatic substrates, is encoded by three genes . One of the gene products, the alkane hydroxylase AlkB, is an integral cytoplasmic membrane protein . Induction leads to the synthesis of 1.5-2% AlkB relative to the total cell protein, both in P . oleovorans and in recombinant Escherichia coli DH1 . We present a study on the induction and localization of the alkane hydroxylase in E . coli W3110, which appears to be an interesting host strain because it permits expression levels of AlkB of up to 10-15% of the total cell protein . This expression level had negative effects on cell growth . The phospholipid content of such cells was about threefold higher than that of wild-type W3110 . Freeze-fracture electron microscopy showed that induction of the alk genes led to the appearance of membrane vesicles in the cytoplasm; these occurred much more frequently in cells expressing alkB than in the negative control, which contained all of the alk genes except for alkB . Isolation and separation of the membranes of cells expressing alkB by density gradient centrifugation showed the customary cytoplasmic and outer membranes, as well as a low-density membrane fraction . This additional fraction was highly enriched in AlkB, as shown both by SDS-PAGE and enzyme activity measurements . A typical cytoplasmic membrane protein, NADH oxidase, was absent from the low-density membrane fraction . alkB expression in W3110 changed the composition of the phospholipid headgroup in the membrane, as well as the fatty acid composition of the membrane . The major changes occurred in the unsaturated fatty acids: C16:1 and C18:1 increased at the expense of C17:0cyc and C19:0cyc.

Protein Eng, 1993 Jun, 6(4), 433 - 40
An EGF-pseudomonas exotoxin A recombinant protein with a deletion in toxin binding domain specifically kills EGF receptor bearing cells; Lee CH et al.; We constructed two chimeric toxins; one composed of epidermal growth factor (EGF) and pseudomonas exotoxin A (PE), designated EGF-PE and the other composed of EGF and PE with a deletion of the Ia domain (cell-binding domain), designated EGF-PE (delta Ia) . Both chimeric toxins reacted with anti-EGF and anti-PE antibodies . The cell-killing experiments showed that EGF-PE, but not EGF-PE (delta Ia), was cytotoxic to the murine fibroblast cell line NR6, which carried the PE receptor, but not the EGF receptor . However, after NR6 was transfected with DNA for the expression of human EGF receptor, the transfected cell line, designated NRHER5, over-expressed human EGF receptors and became sensitive to EGF-PE(delta IA) . The cytotoxicity of EGF-PE(delta Ia), but not EGF-PE, to NRHER5 can be completely blocked by an excess amount of EGF . To completely reverse the cytotoxicity of EGF-PE on NRHER5, both the EGF receptor pathway and the PE receptor pathway need to be blocked . These results suggest that EGF-PE exhibits both EGF and PE binding activities, while EGF-PE(delta IA) possesses only EGF binding activity . Thus, EGF-PE(delta Ia) may be a better chimeric toxin than EGF-PE in terms of target specificity to EGF receptor bearing cells . We, therefore, examined the cytotoxicity of EGF-PE(delta Ia) to various human cancer cell lines . We find that human cancer cells containing more EGF receptors are more sensitive to EGF-PE(delta Ia).

Kidney Int Suppl, 1993 Jun, 41, S205 - 8
Do cytokine-inducing substances penetrate through dialysis membranes and stimulate monocytes?
Kumano K, Yokota S, Nanbu M, Sakai T.
Does a relatively low concentration of endotoxin in dialysate, seen in our clinical dialysis, enhance cytokine production of monocytes across high-flux membranes? Several investigators using extremely high concentrations of endotoxin in dialysate maintain that it does . In vitro experiments in this study were conducted to clarify this . Peripheral blood monocytes were isolated from healthy volunteers and incubated for 20 hours . The incubation medium was made of back-filtrates obtained either from Pseudomonas-contaminated water with endotoxin concentration of 621 pg/ml or water with 10 ng/ml or 1 microgram/ml of lipopolysaccharide (LPS), by passing it through a high-flux membrane dialyzer . Endotoxin free water and addition of LPS (0.01 to 10 ng/ml) were used as a negative and positive control . Interleukin-1 beta (IL-1 beta), IL-6 and tumor necrosis factor alpha (TNF alpha) were measured . Back-filtrate from Pseudomonas-contaminated water did not enhance cytokine production, while 50 pg/ml of endotoxin in culture medium induced a significant cytokine production . Back-filtrate of 1 microgram/ml of endotoxin solution marginally increased IL-6 production, but not the other two cytokines . However, none of the cytokines was induced by the back-filtrate of 10 ng/ml of endotoxin . Monocytes isolated from blood following three hour extracorporeal recirculation did not alter the production of cytokines . These results cannot confirm the transfer of cytokine inducing substances across the membrane at a relatively low endotoxin concentration in dialysate . Further study should be made as to the minimal requirement of dialysate purification for preventing monocyte stimulation.

Biosci Biotechnol Biochem, 1993 Jun, 57(6), 957 - 60
Molecular cloning and nucleotide sequence of the pectate lyase gene from Pseudomonas marginalis N6301; Nikaidou N et al.; Pectate lyase was purified approximately 29-fold to electrophoretic homogeneity from Pseudomonas marginalis N6301 . A pectate lyase (PL; EC4.2.2.2) gene of the strain was cloned and expressed in Escherichia coli . The nucleotides of the PL gene (pel) were sequenced . An open reading frame that encodes a polypeptide (molecular weight: 40,812) composed of 380 amino acids including a 29 amino acid signal peptide was assigned . The structural gene of pel consisted of 1140 base pairs . The nucleotide sequence of the 5'-flanking region of pel showed a consensus sequence of the promoter region of the pectin lyase gene (pnl) in P . marginalis N6301, a Pribnow box, and a ribosome binding site as found in E . coli.

Biosci Biotechnol Biochem, 1993 Jun, 57(6), 931 - 4
Structural and enzymatical comparison of lignostilbene-alpha,beta-dioxygenase isozymes, I, II, and III, from Pseudomonas paucimobilis TMY1009; Kamoda S et al.; Three isozymes of lignostilbene-alpha,beta-dioxygenase (LSD) from Pseudomonas paucimobilis TMY1009 were separated on QAE-Toyopearl chromatography . All active fractions were further chromatographed on DEAE-Toyopearl, Butyl-Toyopearl, and Sephacryl S-300 columns . Then the isozymes I, II, and III were purified homogeneously . All three isozymes consisted of two subunits with the same mol . mass . According to the N-terminal amino acid sequences up to 25 residues of these three isozymes and the reversed-phase HPLC patterns of peptidase-digested them, it was found that LSD-I, II, and III consisted of alpha alpha, alpha beta, and beta beta subunits, respectively . They showed different specificities for several substrates that are stilbene and styrene derivatives.

Biosci Biotechnol Biochem, 1993 Jun, 57(6), 926 - 30
Cloning, expression, and sequence analysis of a lignostilbene-alpha,beta-dioxygenase gene from Pseudomonas paucimobilis TMY1009; Kamoda S et al.; From the genomic library of Pseudomonas paucimobilis TMY1009 constructed with the cosmid pHC79, an 8-kb BamHI-KpnI fragment encoding lignostilbene-alpha,beta-dioxygenase (LSD) was cloned into pUC19 (designated pKSN2510) . E . coli JM109 having pKSN2510 produced a small amount of LSD only when the lac promoter was induced by isopropyl-beta-D-thio-galactopyranoside (IPTG) . The 1.9-kb SalI fragment containing the LSD gene on pKSN2510 was subcloned into pUC119 in opposite directions (designated pKHN2560 and pKHN2590) . The LSD gene on pKHN2590 was expressed in E . coli MV1184 using the lac promoter . LSD produced by E . coli MV1184 transformed with pKHN2590 (cloned LSD) was purified and found to be identical with LSD-I, which was the major isozyme produced by P . paucimobilis TMY1009 . Cloned 1.9-kb nucleotide was sequenced and one open reading frame composed to 486 codons was found.

Mikrobiol Z, 1993 Jun-Aug, 55(4), 43 - 7
{Spontaneous phage production in Pseudomonas pseudomallei and in a range of hosts of melioidosis phages among representatives in the genus Pseudomonas}; Grishkina TA et al.; A capacity to spontaneous phage production has been studied in 57 melioidosis museum strains . The strains were comparatively analyzed by the levels of frequencies of spontaneous phage production . It is established that 40 of 57 studied strains of Pseudomonas pseudomallei are capable to spontaneously produce phages with frequency from 10(7) to 1 . On the indicator lawn of P . mallei phages formed negative colonies of different morphology transparent, turbid and turbid with secondary growth in the centre . No distinct correlation between the frequency of spontaneous phage production and morphology of negative colonies was revealed . The range of hosts of melioidosis phages was determined among 88 representatives of Pseudomonas genus . All melioidosis strains were resistant to their own phages . Phages, identical by the spectrum of lithic effect, were not found even in the case of quantitative coincidence of sensitive strains . Maximum activity was observed in 8 lysates which were lysed 42 to 53 strains of 88 under study.

Chest, 1993 Jun, 103(6), 1706 - 9
Clinical features of Pseudomonas cepacia pneumonia in an epidemic among immunocompromised patients; Yamagishi Y et al.; Between January 1990 and August 1991, there were 37 patients admitted to our Department of Internal Medicine with hematologic malignancies or solid tumors who showed colonization of the respiratory tract with Pseudomonas cepacia . Extensive surveillance cultures of the environmental surfaces and respiratory equipment of the hospital revealed that all nebulizing devices were contaminated with P cepacia . To characterize this outbreak, we retrospectively reviewed the medical records of 37 patients colonized with this organism . All had used nebulizers to deliver aerosols containing polymyxin B and amphotericin B as prophylaxis against infection . Sixteen of these 37 patients developed pneumonia, which was caused in 14 by P cepacia . The majority of the 14 patients showed lobular infiltrates on chest x-ray films . Cavity formation and pleural effusion were observed in 4 of the 14 (29 percent) . All strains of P cepacia were resistant to piperacillin, cefotiam, sulbactam/cefoperazone, moxalactam (latamoxef), cefuzonam, amikacin, tobramycin, ofloxacin, imipenem, and carumonam . Ceftazidime was effective against 84.7 percent of the strains, while minocycline was effective against 63.5 percent of the strains . This appears to be the first report to describe the clinical features of an epidemic of nosocomial P cepacia pneumonia in immunocompromised patients.

J Clin Microbiol, 1993 Jun, 31(6), 1592 - 6
Relationships among Pseudomonas pseudomallei isolates from patients with recurrent melioidosis; Desmarchelier PM et al.; Patients with melioidosis may present with recurrent infections after clinical resolution of their primary illness . Because there has been no satisfactory typing scheme for Pseudomonas pseudomallei, recrudescence could not be distinguished from reinfection . We determined the strain identity of primary and relapse isolates of P . pseudomallei from 25 patients with culture-proven melioidosis to answer whether secondary infections were due to the initial infecting strain or to the acquisition of a new strain . Fifty-four isolates were compared by the patterns of BamHI restriction digests produced after hybridization with a cDNA copy of Escherichia coli rRNA . Twenty-three patients had primary and relapse isolates with identical or highly similar ribotype patterns . The patterns of isolates from two patients were different; the primary and relapse isolates differed by a single fragment for one, and the other had identical primary and first-relapse isolates while the second-relapse isolate was markedly different . The results indicated that recurrent infection probably resulted from endogenous relapse in most of the melioidosis patients studied, although reinfection from an exogenous source was also possible in two cases.

Biochem Biophys Res Commun, 1993 May 28, 193(1), 67 - 76
Isolation and characterization of (+)-1,1a-dihydroxy-1-hydrofluoren-9-one formed by angular dioxygenation in the bacterial catabolism of fluorene; Selifonov SA et al.; Transformation of fluorene by washed cells of fluorene-grown Pseudomonas sp . F274 yielded 1,la-dihydroxy-1-hydrofluoren-9-one (up to 100 mg/l) as the stable product of angular dioxygenation of 9-fluorenone . Structural identity of the angular keto-diol was established by 13C- and 1H-NMR, gas chromatography- and direct probe-mass spectrometry . Definitive assignment of 1,1a-dioxygenation, but not 4,4a-, was based on the isolation and rigorous identification of 1-hydroxyfluoren-9-one as the exclusive product of acidic dehydration . Chiral 1H-NMR analysis and optical rotation of isolated 1,1a-dihydroxy-1-hydrofluoren-9-one ({alpha}D = + 132.1 degrees) are indicative of a single enantiomer with an inferred cis-stereochemistry of the hydroxyl groups . This compound is evidently an intermediate of fluorene catabolism by this strain and not a dead-end product because its formation is transient in washed cell incubations and ultimately it is completely consumed with the formation of acidic metabolites.

Biochem J, 1993 May 15, 292 ( Pt 1), 69 - 74
Protein engineering of the 2-haloacid halidohydrolase IVa from Pseudomonas cepacia MBA4; Asmara W et al.; The chemical modification of L-2-haloacid halidohydrolase IVa (Hdl IVa), originally identified in Pseudomonas cepacia MBA4, produced as a recombinant protein in Escherichia coli DH5 alpha, led to the identification of histidine and arginine as amino acid residues likely to play a part in the catalytic mechanism of the enzyme . These results, together with DNA sequence and analyses {Murdiyatmo, Asmara, Baines, Bull and Hardman (1992) Biochem . J . 284, 87-93} provided the basis for the rational design of a series of random- and site-directed-mutagenesis experiments of the Hdl IVa structural gene (hdl IVa) . Subsequent apparent kinetic analyses of purified mutant enzymes identified His-20 and Arg-42 as the key residues in the activity of this halidohydrolase . It is also proposed that Asp-18 is implicated in the functioning of the enzyme, possibly by positioning the correct tautomer of His-20 for catalysis in the enzyme-substrate complex and stabilizing the protonated form of His-20 in the transition-state complex . Comparison of conserved amino acid sequences between the Hdl IVa and other halidohydrolases suggests that L-2-haloacid halidohydrolases contain conserved amino acid sequences that are not found in halidohydrolases active towards both D- and L-2-monochloropropionate.

Biochemistry, 1993 May 11, 32(18), 4820 - 5
Magnetic circular dichroism studies on the mononuclear ferrous active site of phthalate dioxygenase from Pseudomonas cepacia show a change of ligation state on substrate binding; Gassner GT et al.; Phthalate dioxygenase from Pseudomonas cepacia contains a mononuclear ferrous center that is strictly required for catalytic oxygen activation . The spectroscopic characterization of this iron site and its ligand interactions has been complicated in the past by interference from a Rieske-type binuclear (2Fe-2S) cluster in the enzyme, which dominates the absorption spectra and is superimposed in X-ray absorption spectra for the mononuclear site . We have used low-temperature, variable magnetic field circular dichroism spectroscopy to selectively detect the ligand field spectra of the paramagnetic mononuclear ferrous active site in the presence of the diamagnetic exchange-coupled Rieske center and observe spectral changes associated with substrate binding . The perturbations of the d-->d spectra for the mononuclear ferrous site reflect a decrease in coordination number from six to five on binding substrate . This structural change suggests that displacement of an iron ligand prepares the ferrous center for dioxygen activation.

Biochemistry, 1993 May 4, 32(17), 4552 - 9
Transient kinetics of electron transfer from a variety of c-type cytochromes to plastocyanin; Meyer TE et al.; Plastocyanin (PC) and its physiological reaction partner cytochrome (cyt) f form a complex which is electrostatically stabilized by interactions between complementary localized charges . We have measured the kinetics of intracomplex electron transfer between several reduced cytochromes and PC using laser flash photolysis . With spinach cyt f and spinach PC, we obtain first-order rate constants, kforward = 2780 s-1 and kreverse = 1050 s-1, for the reversible reaction and a complex dissociation constant of about 23 microM at an ionic strength (I) of 5 mM . The observed rate constant increases by a factor of 2 between I = 5 and 40 mM and then decreases monotonically at higher ionic strengths . This indicates that the complex is not completely dissociated until I = 150 mM and that the proteins within the electrostatically most stable complex are not optimally oriented for electron transfer . Similar results were obtained with turnip cyt f and spinach PC, although in this case intracomplex electron transfer is about 4 times as fast . Horse cyt c also forms an electrostatically stabilized complex with PC, and yields a limiting rate constant for intracomplex electron transfer (1750 s-1) and a dissociation constant (10 microM) comparable to those for spinach cyt f . The ionic strength dependence shows that the complex is more readily dissociated (complete at I = 25 mM) than is that of cyt f and that rearrangement is not required for optimal electron transfer . Addition of polylysine results in 10-fold inhibition of the rate of electron transfer . Pseudomonas cyt c-551 is an acidic cytochrome which does not form a complex with PC.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1993 May, 59(5), 1403 - 9
Bromoalkane-degrading Pseudomonas strains; Shochat E et al.; Two Pseudomonas isolates, named ES-1 and ES-2, were shown to possess a wide degradative spectrum for haloalkanes in general and bromoalkanes in particular but did not degrade nonsubstituted alkanes . The utilization of water-insoluble haloalkanes, such as 1-bromooctane, appeared to consist of three phases: (i) extracellular emulsification by a constitutively excreted, broad-spectrum surface-active agent, (ii) dehalogenation by an inducible hydrolytic dehalogenase (possibly periplasmic), and (iii) intracellular degradation of the residual carbon skeleton . Several observations suggest the existence of more than one dehalogenase in strain ES-2.

Mol Gen Genet, 1993 May, 239(1-2), 6 - 16
Molecular characterization and hrp dependence of the avirulence gene avrPto from Pseudomonas syringae pv . tomato {corrected}; Salmeron JM et al.; The avrPto avirulence gene from Pseudomonas syringae pv . tomato (Pst) race 0 governs race-specific resistance to bacterial speck disease in tomato cultivars containing the Pto resistance gene . The avrPto gene encodes 0.7 and 0.75 kb mRNAs whose predicted translation product is a mostly hydrophilic 164 amino acid protein of 18.3 kD a that reveals no homology to protein sequences in GenBank or EMBL databases . Highest expression of avrPto in cell culture is observed in minimal media containing sugars and sugar alcohols as carbon sources and lowest expression in minimal media containing tricarboxylic acid intermediates and in complex media . Expression of avrPto in planta is induced within 1 h following infection of both resistant and susceptible tomato plants by Pst, and increases over the first 6 h . Transcription of avrPto requires the hrpSR pathogenicity functions, but is independent of other Pst hrp genes . A region of the avrPto promoter shows homology to hrp box sequences upstream of other P . syringae genes that require the hrpSR locus for expression, and both avirulence activity and avrPto mRNA accumulation are abolished by deletions extending into this region . The avrPto transcription start site maps 31 nucleotides downstream of the hrp box motif.

Mol Gen Genet, 1993 May, 239(1-2), 17 - 27
Genetic characterization of the Pto locus of tomato: semi-dominance and cosegregation of resistance to Pseudomonas syringae pathovar tomato and sensitivity to the insecticide Fenthion; Carland FM et al.; The Pto locus governs resistance to bacterial speck disease in tomato caused by race 0 strains of Pseudomonas syringae pathovar tomato (Pst) . Large populations segregating for the Pto locus were generated and genetically characterized . Analysis of the locus has revealed that Pto acts in a semi-dominant manner and cosegegrates with sensitivity to an organophosphorous insecticide, Fenthion, suggesting that Pto may be a complex locus responsible for both phenotypes . We have redefined its map position on chromosome five of the classical genetic map and assigned its position on the molecular map, thus facilitating the alignment of the two genetic maps of the short arm of chromosome five of tomato . Furthermore, we have screened random amplified polymorphic (RAPD) markers for their ability to differentiate near-isogenic lines that differ only with respect to Pto and have identified and mapped seven of these markers . Our results suggest that Pto may be located in a euchromatic region on chromosome five which will be advantageous for the cloning of this locus by one of several molecular strategies.

Eur J Biochem, 1993 May 1, 213(3), 1081 - 9
Human 4-hydroxyphenylpyruvate dioxygenase . Primary structure and chromosomal localization of the gene; Ruetschi U et al.; We report the primary structure of 4-hydroxyphenylpyruvate dioxygenase {4-hydroxyphenyl-pyruvate:oxygen oxidoreductase (hydroxylating, decarboxylating)} . The work is based on the isolation of cDNA clones from human liver lambda gt11 libraries . Several overlapping clones covering the coding sequence were characterized . In parallel, peptides from four different digests of the purified protein were analysed for their amino-acid sequence . These peptide sequences covered 86% of the cDNA-derived amino-acid sequence . This gives the sequence for a polypeptide of 392 amino acids with a calculated molecular mass of 44.8 kDa . There is more than 80% identity between the human and the pig enzymes and also between these enzymes and the F antigen from rat and the two allelic forms of this antigen from mouse . The enzyme has 53% conserved amino acids and 27% identical amino acids in common with 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp . P.J . 874 and 52% conserved and 28% identical residues, with a protein from Shewanella colwelliana . At the C-terminus there is 61% identity between the seven proteins . These results indicate that these proteins are all 4-hydroxyphenylpyruvate dioxygenases . The identity of the C-terminus makes this part of the molecule a candidate for a functional role in the catalytic process . At conserved positions in all seven enzymes, there are two tyrosine residues and three histidine residues, i.e . amino acids which have been implicated as ligands for iron in 2-oxoacid-dependent dioxygenases . The gene encoding the enzyme was localized to chromosome 12q14-->qter by Southern-blot analysis of human-rodent somatic-cell hybrids.

Eur J Biochem, 1993 May 1, 213(3), 1075 - 80
gamma-Butyrobetaine hydroxylase . Structural characterization of the Pseudomonas enzyme; Ruetschi U et al.; gamma-Butyrobetaine hydroxylase is a 2-oxoglutarate-dependent dioxygenase that catalyzes the hydroxylation of gamma-butyrobetaine to carnitine, the last step in the biosynthesis of carnitine from lysine . The primary structure of the enzyme from Pseudomonas sp . AK1 has been determined . Sequence analysis of the intact protein and of peptides from essentially three different digests established the presence of a peptide chain containing 383 residues, and an N-terminal truncated form of 382 residues . The two chains have molecular masses of 43,321 Da and 43,207 Da, respectively, and are identical except for the presence or absence of an N-terminal asparagine residue; the shorter form starts with an alanine residue . In preparations of the dimeric protein, the two chains occur in an approximate ratio of 1:1 . There are nine cysteine residues and 13 histidine residues, i.e . amino acids which have been postulated as ligands for iron binding . In spite of functional similarities, there appears to be no clear sequence similarities with any of the other mammalian 2-oxoglutarate-dependent dioxygenases so far characterized.

Ann Thorac Surg, 1993 May, 55(5), 1087 - 91; discussion 1091-2
Double-lung transplantation in mechanically ventilated patients with cystic fibrosis; Massard G et al.; Many lung transplant programs consider ventilator dependence as a contraindication for transplantation . Among 54 patients in whom bilateral lung transplantations for cystic fibrosis were performed by the Joint Marseille-Montreal Lung Transplant Program, 10 were ventilator dependent . Three of them died in the early postoperative period (30%): 2 as a result of cerebral anoxia and sepsis, 1 of Pseudomonas cepacia pneumonia . Two patients died at 15 and 19 months after transplantation of obliterative bronchiolitis and secondary bacterial pneumonitis . Another 2 patients in whom obliterative bronchiolitis developed underwent retransplantation with a heart-lung block; 1 of those was operated on at 12 months and is well at 29 months after his initial transplantation; the second was operated on at 34 months and died of primary graft failure . Three other patients are alive and well at 3, 11, and 14 months after transplantation . Actuarial survival at 1 year was 70% . The postoperative course and the infectious and rejection complications were no different from those in patients who underwent transplantation while spontaneously breathing . Obliterative bronchiolitis developed in 66% of patients at risk (2 of 6 patients surviving more than 6 months) . We conclude that transplantation in mechanically ventilated patients with cystic fibrosis is not associated with an increase in morbidity or mortality after bilateral lung transplantation . Long-term survival, as in patients who undergo transplantation while spontaneously breathing, is limited by the development of obliterative bronchiolitis.

Biochem J, 1993 May 1, 291 ( Pt 3), 889 - 94
N.m.r . spectroscopic studies of fucose-containing oligosaccharides derived from keratanase digestion of articular cartilage keratan sulphates . Influence of fucose residues on keratanase cleavage; Tai GH et al.; Keratan sulphate chains from bovine articular cartilage were fully digested with keratanase from Pseudomonas sp . and the products were reduced with alkaline borohydride . The resultant fragments were fractionated on a Nucleosil 5SB column and the earliest eluting fucose-containing oligosaccharides were isolated . Structural analysis using 1H n.m.r . spectroscopy (600 MHz) showed the two least-charged species to have the following structure: GlcNAc(6S) beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc(6S) beta 1- 3Gal beta 1-4GlcNAc(6S) beta 1-3Gal-ol and GlcNAc(6S) beta 1-3Gal beta 1- 4(Fuc alpha 1-3)GlcNAc(6S) beta 1-3Gal beta 1-4GlcNAc(6S) beta 1-6(Gal beta 1- 3)GalNAc-ol . Both galactoses adjacent to the fucosylated N-acetylglucosamine residue are unsulphated . Therefore, it can be deduced from these structures that the presence of fucose on N-acetylglucosamine residues in keratan sulphates protects both of the adjacent unsulphated galactose residues from keratanase cleavage . This result has implications for the interpretation of keratanase fingerprints, because in articular cartilage keratan sulphates the keratanase-resistant blocks are not solely those with fully sulphated galactose residues, but also include the fucosylated sequences, which have unsulphated galactoses . It is, therefore, not possible to estimate their galactose sulphation or the size of the fully sulphated disaccharide-repeat sequences from keratan sulphates that contain fucose.

J Gen Microbiol, 1993 May, 139 ( Pt 5), 1019 - 25
Bacterial metabolism of 5-aminosalicylic acid: enzymic conversion to L-malate, pyruvate and ammonia; Stolz A et al.; 5-Aminosalicylate (5AS) was converted to L-malate, pyruvate and ammonia by cell-free extracts from Pseudomonas sp . BN9 in the presence of glutathione . In the absence of glutathione, 5AS was oxidized to the ring-fission product cis-4-amino-6-carboxy-2-oxo-hexa-3,5-dienoate (cis-ACOHDA) . Glutathione catalysed the spontaneous isomerization of cis-ACOHDA to its trans-isomer . The same reaction was catalysed by light and by acidic conditions . trans-ACOHDA was enzymically deaminated to fumarylpyruvate (trans-2,4-dioxo-5-hexenoate) . The trans-ACOHDA hydrolase was induced after growth of Pseudomonas sp . BN9 with 5AS, but not after growth with acetate or nutrient broth . At the fumarylpyruvate stage, the metabolism of 5AS converged with a pathway described for the degradation of gentisate . Fumarylpyruvate was cleaved by Pseudomonas sp . BN9 to fumarate and pyruvate.

Mol Microbiol, 1993 May, 8(4), 643 - 52
Identification of a gene cluster encoding three high-molecular-weight proteins, which is required for synthesis of tolaasin by the mushroom pathogen Pseudomonas tolaasii; Rainey PB et al.; The extracellular lipodepsipeptide toxin tolaasin is the primary disease determinant of pathogenicity of Pseudomonas tolaasii on the cultivated mushroom, Agaricus bisporus . Transposon mutagenesis of P . tolaasii NCPPB 1116 with Tn5-generated 5000 chromosomal insertions of which 35 (0.7%) were tolaasin negative and 12 (0.25%) produced a reduced amount of tolaasin . In addition, TnphoA mutagenesis yielded a single tolaasin-negative mutant which was phoA active . Restriction enzyme mapping of mutant DNAs by Southern hybridization analysis revealed that the majority of Tn5 insertions were confined to a single genetic locus of approximately 65 kbp . Pulsed-field gel electrophoresis of representative Tn5 mutant DNAs showed that this region is at one end of a 640 kbp PacI chromosomal fragment and that the P . tolaasii genome is 6.7 Mbp . SDS-PAGE analysis of protein extracts from wild-type P . tolaasii demonstrated the presence of three high-molecular-weight proteins (designated TL1, TL2 and TL3) . Alterations in the presence of these proteins, as well as apparently truncated forms of the 465 kDa (TL1), 440 kDa (TL2) and 435 kDa (TL3) proteins were observed in some mutants, enabling the direction and order of the transcriptional units to be determined . Two other Tn5 mutations were also identified which resulted in a tolaasin-negative phenotype, but which did not affect the expression of TL1, TL2, or TL3 . One of these mutants is linked to the TL-cluster, but the other is located outside this region . It is concluded that at least five genetic loci, including those encoding TL1, TL2 and TL3, are required for tolaasin synthesis.

Mol Microbiol, 1993 May, 8(3), 625 - 35
Multiple copies of a DNA sequence from Pseudomonas syringae pathovar phaseolicola abolish thermoregulation of phaseolotoxin production; Rowley KB et al.; Phaseolotoxin, a phytotoxin of Pseudomonas syringae pv . phaseolicola, is produced at 18 degrees C but not at 28 degrees C . Here we report that a fragment (24.4 kb) cloned from the wild-type strain, which does not harbour a gene(s) involved in phaseolotoxin biosynthesis, abolishes this thermoregulation in the wild type and suppresses a Tox- mutant at both temperatures . A subclone harbouring a 485 bp fragment contains motifs that are characteristic of DNA-binding sites . In mobility shift assays we have detected a protein(s) from the wild-type and the mutant strains, grown at appropriate temperatures, that specifically binds to the fragment containing the DNA-binding motifs . We propose that the binding protein is a repressor which is 'titrated' by this fragment when it is present in the cell on a multiple copy plasmid, thus allowing expression of phaseolotoxin genes.

Zhonghua Yu Fang Yi Xue Za Zhi, 1993 May, 27(3), 144 - 6
{Study on distribution of the Pseadomonas cocovenenans subsp farinofermentans Meng ZH&Wang DS et al in the natural environment of Hebei Province}; Hou ZZ; In this paper, the distribution of the Pseudomonas Cocovenenans Subsp Farinofermentans Meng ZH&Wang DS et al in the natural environment of Hebei Province is reported . Several food poisoning cases caused by the bacterium have taken place, but it is still unusual in the natural environment such as soid and food . Fresh tremella and mushroom are heavily polluted by the bacteria . New knowledge is gained in the differential diagnosis between the poisoning caused by poisonous mushroom and this bacteria . Control measures are suggested for the prevention and cure of the food poisoning caused by this bacteria.

Biotechnol Prog, 1993 May-Jun, 9(3), 323 - 31
Molecular cloning of the gamma-glutamyltranspeptidase gene from a Pseudomonas strain; Ishiye M et al.; gamma-Glutamyltranspeptidase (GGT) was purified from a Pseudomonas sp . strain A14 . The purified enzyme was found to be composed of two nonidentical subunits with molecular weights of 39,000 and 22,000 and had a pI of > 8.6 . The partial N-terminal amino acid sequences of both subunits and some proteolytic fragments were determined . Using mixed oligonucleotides designed from the partial amino acid sequences as hybridization probes, one cosmid clone which contained the GGT gene was isolated from a Pseudomonas sp . strain A14 cosmid genome library, and the DNA sequence of the GGT gene was determined . The nucleotide sequence and the protein sequence analysis revealed that GGT was synthesized as a precursor protein of 575 amino acids and then processed to mature enzyme, presumably after removal of a signal peptide . Comparison of the predicted amino acid sequence of Pseudomonas GGT with published results for Escherichia coli K-12 and rat kidney GGTs shows that the protein sequence of Pseudomonas GGT is 51% and 33% identical to the E . coli and rat GGT sequences, respectively . Higher similarity is observed among the small subunits, which have been thought to have a binding site for the gamma-glutamyl residue . Expression of the cloned Pseudomonas GGT gene in E . coli was subjected to Western blot analysis using antibody raised against the purified GGT . This suggested that processing of the precursor protein to its subunits is temperature-dependent, because the amount of mature GGT protein was increased when the culture was performed at low temperature.

Biosci Biotechnol Biochem, 1993 May, 57(5), 703 - 9
Isolation and characterization of Tn5-induced mutants of Pseudomonas paucimobilis UT26 defective in gamma-hexachlorocyclohexane dehydrochlorinase (LinA); Nagata Y et al.; Pseudomonas paucimobilis UT26 grows on gamma-hexachlorocyclohexane (gamma-HCH) as a sole source of carbon and energy . Tn5 mutation was introduced into UT26, and two kinds of mutants defective in gamma-HCH degradation were phenotypically isolated; one (UT64) completely lacked the activity to degrade gamma-HCH, while the other (UT61) retained a very low level of activity . Tagging and sequencing analysis showed that both mutants had a Tn5 insertion at the same site of the linA (gamma-HCH dehydrochlorinase encoding) gene . However, UT61 had an additional rearrangement, which could be the cause of its retaining a low level of activity . An in vitro complementation test with a crude extract from UT64 plus partially purified LinA protein showed that LinA was essential not only for the first-step reaction (gamma-HCH to gamma-pentachloro-cyclohexene; gamma-PCCH), but also for the second-step reaction (gamma-PCCH to compound B) of gamma-HCH degradation in UT26.

J Bacteriol, 1993 May, 175(10), 2994 - 3001
Dienelactone hydrolase from Pseudomonas cepacia; Schlomann M et al.; Dienelactone hydrolases have previously been shown to play a crucial role in chlorocatechol degradation via the modified ortho cleavage pathway . Recently, the enzymes induced in 4-fluorobenzoate-utilizing bacteria have been classified into three groups on the basis of their specificity towards cis- and trans-dienelactone . The dienelactone hydrolase and the 3-oxoadipate enol-lactone hydrolase from Pseudomonas cepacia have now been purified to apparent homogeneity and characterized with respect to molecular mass and amino acid composition . The dienelactone hydrolase has a distinct preference for cis-dienelactone and did not convert the trans isomer or muconolactone, 3-oxoadipate enol-lactone, or 4-fluoromuconolactone to a significant extent . In properties like amino acid composition, pH optimum of activity, and lack of inhibition by p-chloromercuribenzoate, the P . cepacia dienelactone hydrolase differed substantially from 3-oxoadipate enol-lactone hydrolases and other dienelactone hydrolases.

Biochem Biophys Res Commun, 1993 Apr 30, 192(2), 976 - 81
Catalytic properties and stability of a Pseudomonas sp.101 formate dehydrogenase mutants containing Cys-255-Ser and Cys-255-Met replacements; Tishkov VI et al.; Two mutants of bacterial formate dehydrogenase from Pseudomonas sp.101 (EC 1.2.1.2, FDH)-C255S (FDH-S) and C255M (FDH-M), were obtained and its properties were studied . Both mutations provided the high resistance to inactivation by Hg2+ . Slow inactivation of mutants by DTNB reveals the presence in FDH molecule of another essential cysteine residue . Specific activities of FDH, FDH-S and FDH-M were 16, 16 and 9.5 U/mg of protein, respectively . Km on formate was 7.5, 7.5 and 20 mM and Km on NAD(+)-0.1, 0.3 and 0.6 mM for FDH, FDH-S and FDH-M, respectively . Mutations of Cys255 on Ser or Met resulted in increasing of enzyme stability at 25 degrees C and decreasing of thermostability (above 45 degrees C) . Data obtained show that Cys255 is unique residue for providing both enzyme thermostability and catalytically optimal binding of coenzyme.

Biochemistry, 1993 Apr 20, 32(15), 4029 - 34
Spectroscopic and kinetic studies of lipases solubilized in reverse micelles; Walde P et al.; The conformation and activity of three different lipases have been studied in reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane . In the case of human pancreatic lipase, the conformation of the polypeptide chain--as judged from far-UV circular dichroism measurements--is only slightly altered after the enzyme is transferred from a bulk aqueous solution into the microenvironment of reverse micelles . Significant spectral changes in the near-UV circular dichroism and fluorescence spectrum indicate, however, that the solvation of aromatic amino acid side chains is considerably different in reverse micelles . Conversely, the circular dichroism spectra of the lipases from Candida rugosa and Pseudomonas sp . are considerably different in reverse micelles, compared with the spectra in aqueous solution, indicating that both enzymes loose the native structure at the water/AOT/oil interface . Bound substrate and/or product can prevent this denaturation . While Pseudomonas sp . and human pancreatic lipase are inhibited by tetrahydrolipstatin (THL), the lipase from Candida rugosa is not . These data, together with additional activity and inhibition data, indicate that the micellar microenvironment accentuates the difference between the different enzymes in terms of the relation structure/activity.

Proc Natl Acad Sci U S A, 1993 Apr 15, 90(8), 3530 - 4
Targeted delivery of peptide epitopes to class I major histocompatibility molecules by a modified Pseudomonas exotoxin; Donnelly JJ et al.; Cytotoxic T lymphocytes (CTLs) expressing the CD8 surface marker recognize peptides in association with major histocompatibility complex (MHC) class I molecules . Although most peptides expressed on MHC class I molecules are derived from self- or virally encoded proteins, delivery of exogenous proteins to the cytosol can result in their being processed for presentation to CTLs on MHC class I molecules . We describe two fusion proteins (PEMa and PENP), consisting of the binding and translocating domains of Pseudomonas exotoxin A (PE), fused to peptide epitopes from influenza A matrix protein and nucleoprotein, respectively . These fusion proteins were internalized and processed by MHC class I-positive target cells, resulting in sensitization of target cells for lysis by peptide-specific CTLs . A point mutation known to interfere with intoxication by wild-type PE also reduced the ability of PEMa to sensitize target cells . Fusion of peptide or polypeptide epitopes with PE provides a potential means of eliciting CTLs without the use of self-replicating agents, as well as a useful probe for studying MHC class I-restricted antigen processing.

Biochemistry, 1993 Apr 6, 32(13), 3488 - 97
A transforming growth factor-alpha-Pseudomonas exotoxin hybrid protein undergoes pH-dependent conformational changes conducive to membrane interaction; Sanyal G et al.; TP40 is a chimeric protein containing transforming growth factor alpha (TGF-alpha) at the N-terminus and a derivative of a 40,000-Da segment (PE40 delta cys) of Pseudomonas exotoxin (PE) . PE40 delta cys contains domains Ib, II, and III of PE in which the cysteines are mutated to alanines . The rationale for inclusion of TGF-alpha is to provide TP40 with selective targeting toward cells expressing the epidermal growth factor receptor (EGFr) on their surface {Pastan, I., & FitzGerald, D . (1989) J . Biol . Chem . 264, 15157-15160} . Translocation across endosomal membranes is thought to be a required step for cytotoxic activity of PE . This step is presumably facilitated by the low pH in endosomes which induces exposure of a hydrophobic surface of the protein, which in turn becomes available to interact with and translocate across the membrane . We have employed the hydrophobic fluorescence probe 2-p-toludinylnaphthalene-6-sulfonate (TNS) and the intrinsic tryptophan fluorophores of TP40 to investigate pH-induced changes in the tertiary structure of this protein . The pH dependence of TP40 interaction with liposomes also provided a model for studying protein-membrane interactions . TNS fluorescence was markedly enhanced in the presence of TP40 below pH 4 and to a lesser degree between pH 7 and 5 . A progressive red shift of tryptophan fluorescence with decreasing pH was also seen with the approximate midpoint for this transition occurring around pH 3 . Both observations suggest that acidic pH induces exposure of hydrophobic regions of TP40, making them accessible to solvent and TNS . No major alteration of the secondary structure was manifested in the far-UV CD spectrum of TP40 upon a reduction in pH from 7 to 2 . Thus, the low-pH-induced structural change of TP40 appears to involve a subtle exposure of one or more hydrophobic surfaces without an extensive unfolding of the protein's secondary structure . In the presence of anionic liposomes, a low-pH-induced blue shift of the TP40 tryptophan fluorescence was observed, suggesting that interaction with liposomes also required the low-pH conformation of the protein . However, the midpoint of this fluorescence blue shift occurred at approximately pH 5, which is presumably closer to the physiological pH within endosomes . Neutral liposomes failed to induce these spectral changes in TP40, implying a lack of interaction with these lipids . At acidic pH values between 2 and 4, self-association of TP40 in solution was detected by equilibrium sedimentation and quasielastic light scattering measurements . This probably results from intermolecular interaction between exposed hydrophobic surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)

J Gen Microbiol, 1993 Apr, 139 ( Pt 4), 743 - 52
Identification of methanol-regulated promoter sequences from the facultative methylotrophic bacterium Methylobacterium organophilum XX; Xu HH et al.; A promoter-probe vector (pHX200) was constructed using the broad-host-range cosmid pLA2917 and a promoterless xylE gene of Pseudomonas as the reporter gene . Insertion of the cloned promoter fragment of the methanol dehydrogenase large subunit gene moxF (methanol oxidation) in front of the xylE gene in pHX200V-47 resulted in high-level expression of the xylE gene product--catechol 2,3-dioxygenase--in Methylobacterium organophilum XX . The specific activity of the enzyme was four times higher in methanol-grown M . organophilum XX culture than in succinate-grown culture . Interestingly, the insertion of the same fragment in the opposite orientation in front of the xylE gene (pHX200V-74) also led to elevated catechol 2,3-dioxygenase activity . This promoter activity was also methanol regulated . A total of 21 methanol-regulated promoter clones were identified that originate from three gene clusters (groups V, VI and VII) on the M . organophilum XX chromosome involved in methanol oxidation . Vector pHX200 and its derivatives were successfully mobilized into cells of three phylogenetically diverse methylotrophic bacteria: Methylophilus methylotrophus AS1, Methylobacterium extorquens AM1 and Methylobacterium sp . DM4 . The reporter gene (xylE) was functionally expressed in all three bacteria with the aid of a proper promoter . Transcriptional fusions of methanol-regulated promoters with the xylE gene were mobilized into Mox- mutants of M . organophilum XX and M . extorquens AM1 to study the roles of methanol oxidation genes, especially regulatory genes . It appeared that vector pHX200 is an efficient promoter probe with wide host-range and an excellent tool for studies of structure and function of promoters/regulators.

FEMS Microbiol Lett, 1993 Apr 1, 108(2), 219 - 24
Degradation of 2,4-dihydroxybenzoate by Pseudomonas sp . BN9; Stolz A et al.; The aerobic degradation of 2,4-dihydroxybenzoate by Pseudomonas sp . BN9 was studied . Intact cells of Pseudomonas sp . BN9 grown with 2,4-dihydroxybenzoate oxidized 2,4-dihydroxybenzoate but not salicylate . Cell-free extracts of Pseudomonas sp . BN9 converted 2,4-dihydroxybenzoate after the addition of NAD(P)H . A partially purified protein fraction converted 2,4-dihydroxybenzoate with NADH to 1,2,4-trihydroxybenzene . 1,2,4-Trihydroxybenzene was converted by a 1,2-dioxygenase to maleylpyruvate, which was reduced by a NADH-dependent enzyme to 3-oxoadipate . 2,4-Dihydroxybenzoate 1-monooxygenase, 1,2,4-trihydroxybenzene 1,2-dioxygenase and maleylpyruvate reductase were induced in Pseudomonas sp . BN9 after growth with 2,4-dihydroxybenzoate.

Appl Environ Microbiol, 1993 Apr, 59(4), 1082 - 91
Dynamics, spread, and persistence of a single genotype of Pseudomonas syringae relative to those of its conspecifics on populations of snap bean leaflets; Hirano SS et al.; A rifampin-resistant strain of Pseudomonas syringae (R10) was introduced onto bean plants grown in field plots to examine the processes of growth, spread, and survival of a single genotype relative to the dynamics of its conspecifics on populations of individual leaflets . R10 was applied to four plots (400, 200, 100, and 50 m2), each of which was centered in a quadrant of a bean field (90 by 90 m) . Population sizes of the species P . syringae and of R10 were determined on each of 25 individual leaflets collected from the largest plot (A) at approximately weekly intervals during a 10-week period following application . The spread of R10 from all plots was monitored by leaf imprinting of individual leaflets collected at sites along four transects, each of which bisected two of the plots . The introduced strain was a dominant component of the species for about 5 weeks postinoculation on leaflets from plot A . Although the population sizes of R10 remained at about 5.0 to 5.5 log10 CFU per leaflet, the strain became a progressively minor component of the species as the population sizes of its conspecifics continued to increase during the latter part of the growing season . In general, a positive correlation was found for the population sizes of R10 and its conspecifics on individual leaflets collected throughout the growing season . This result suggests that large numbers of R10 early in the growing season did not exclude the colonization of bean leaflets by its conspecifics . It is apparent that the species pool comprised genotypes that were more fit than R10 under the conditions that prevailed during the latter part of the growing season . By 6 weeks postinoculation, R10 was detected at all sites sampled within the unsprayed areas of the field . However, it was present as a minor component of the species . The persistence of R10 throughout the winter and into the following growing season was monitored in plot A, which was plowed and replanted with wheat in the fall . R10 was detected on some of the samples (wheat seedlings or soil) taken at approximately monthly intervals from November to June of the following year . In June, the field was plowed and replanted with beans . We could not detect R10 on emerging bean seedlings in plot A . The results demonstrate that the successful spread and persistence of an introduced bacterium do not necessarily lead to the establishment of large populations of the bacterium in adjacent untreated areas or on its host plant in subsequent growing seasons.

Leukemia, 1993 Apr, 7(4), 553 - 62
Cytotoxic activities of recombinant immunotoxins composed of Pseudomonas toxin or diphtheria toxin toward lymphocytes from patients with adult T-cell leukemia; Kreitman RJ et al.; We have previously shown that the recombinant single-chain immunotoxin anti-Tac(Fv)-PE40, composed of the variable domains of the anti-Tac monoclonal antibody in a single-chain form joined to a derivative of Pseudomonas exotoxin (PE), is cytotoxic toward malignant cells from adult T-cell leukemia (ATL) patients . Using this assay, we have now compared the activity of anti-Tac(Fv)-PE40 with that of an improved version, anti-Tac(Fv)-PE40KDEL which contains an altered carboxyl terminus, and also with two chimeric toxins made with diphtheria toxin (DT) . One of these is a fusion of amino acids 1-388 of DT with anti-Tac(Fv) and is termed DT388-anti-Tac(Fv) . The other, DT388-IL2, contains interleukin 2 (IL2) at the carboxyl terminus of the same DT derivative . We incubated these toxins with malignant ATL peripheral blood mononuclear cells (PBMCs) for 1-3 days and then measured {3H}leucine incorporation . We found that anti-Tac(Fv)-PE40KDEL was the most cytotoxic agent and was followed in decreasing order of activity by anti-Tac(Fv)-PE40, DT388-anti-Tac(Fv), and finally DT388-IL2 . Trypan blue staining showed that inhibition of protein synthesis correlated with cell death . Time course studies showed that the recombinant toxins containing anti-Tac(Fv) were cytotoxic even if exposed to the cells for only one hour . After intravenous injection into mice, the half-life of anti-Tac(Fv)-PE40 or anti-Tac(Fv)-PE40KDEL was 30 minutes . Normal PBMCs were resistant to all four toxins . Recombinant immunotoxins made with anti-Tac merit further study as potential reagents in the treatment of ATL.

J Immunol, 1993 Apr 1, 150(7), 2774 - 82
Recombinant immunotoxins containing the VH or VL domain of monoclonal antibody B3 fused to Pseudomonas exotoxin; Brinkmann U et al.; We prepared recombinant immunotoxins in Escherichia coli in which the VH or VL domains of mAb B3 were fused to a truncated form of Pseudomonas exotoxin (PE) (PE38KDEL) . mAb B3 binds to a carbohydrate Ag found on the surfaces of many types of cancers and only a few normal tissues . PE38KDEL is a 38-kDa form of PE (66 kDa) that is missing the cell-binding domain of PE and has the carboxyl end changed from REDLK to KDEL . We show that immunotoxins in which the H chain or the L chain V region is fused to PE38KDEL bind to and kill carcinoma cells containing the B3 Ag . B3 Ag-negative cells were not affected . The cytotoxicity of these molecules is between 20- and 100-fold less than B3(Fv)-immunotoxins, containing both the H and L chain V regions . The VL-containing toxin was more active than the VH-containing toxin, indicating that the L chain of mAb B3 probably contributes more to Ag-binding than the H chain . Refolding experiments show that B3(VL)-PE38KDEL aggregates less than the VH-derivative or than a single chain immunotoxin B3(Fv)-PE38KDEL, which contains both domains in a single chain form . Furthermore, in addition to monomers, active homodimers of B3(VH)- and B3(VL)-PE38KDEL were obtained from renaturation experiments . The VL-toxin dimers, which might have their binding regions arranged in a manner similar to Bence Jones proteins (L chain homodimers), were found to have almost the same cytotoxicity as the monomers, whereas the VH-toxin dimers had decreased cytotoxic activity.

Rev Argent Microbiol, 1993 Apr-Jun, 25(2), 70 - 9
{In vitro control of Sclerotinia sclerotiorum and Gaeumannomyces graminis by bacteria of the fluorescent Pseudomonas group}; Andreoli YE et al.; Thirty six fluorescent Pseudomonas isolates were obtained from the rhizosphere of sunflower plants . By antibiosis tests, the six more efficient strains in Sclerotinia sclerotiorum growth inhibition, were selected . Simultaneously, twenty three fluorescent Pseudomonas isolates were recuperated from the rhizosphere of wheat plants and the five most efficient strains in growth inhibition of the fungi Gaeumannomyces graminis were selected . The strains selected from the rhizosphere of sunflower plants had no antagonistic effect on G . graminis and the bacteria isolated from the wheat rhizosphere showed no fungistatic activity on S . sclerotiorum . These results suggest the existence of a certain degree of plant bacteria pathogenic specificity . Among the selected bacteria, the strain FF5 of P . fluorescens originated the major inhibiting halo in vitro against S . sclerotiorum (Figure 1) . In liquid culture medium this bacterium produces an antifungal substance that promotes lysis of fungi mycelium (Figure 2) and inhibition of ascospore germination and is not inhibited by the presence of Fe+3 in the culture medium (Table 1) . Its synthesis is not associated with the production of fluorescein . Its action is not enzymatic because it is a substance of low molecular weight (< 2000), resistant to autoclave sterilization and photo-stable . The amount of NH4+ and the high pH values produced by the FF5 strain in the liquid culture medium (Table 2) are not responsible for the antifungalal action.

Respir Med, 1993 Apr, 87(3), 187 - 92
Pseudomonas cepacia: pulmonary infection in patients with cystic fibrosis; Taylor RF et al.; This retrospective study reviews the patterns of P . cepacia pulmonary infection in 75 of a total of 872 mainly adults with cystic fibrosis, registered here during the 4 years 1987-1990; 35 (47%) were female . During this period, 55 patients acquired P . cepacia and the annual incidence and prevalence rates have remained between 1.6 and 3.1%, and 4.1 and 5.9%, respectively . The mean age at the time of the first isolation of P . cepacia was 23 years, ranging 11-45 years . Sixty-eight percent of the initial isolates were multi-resistant (sensitive to fewer than three of 15 anti-pseudomonal agents) . Prior to acquisition of P . cepacia, 28 (50.9%) patients already had severe lung disease and only three had normal lung function . Infection was transient in 39.1% of patients . Initial multi-resistance of P . cepacia to anti-pseudomonal agents was significantly associated with persistent infection . Clinical outcome was unaffected by age, sex and early intravenous antibiotic therapy but was significantly adversely affected by increasing severity of lung disease at the onset of P . cepacia infection and by initial multi-resistance, and persistence of the organism . Thus, all patients with normal or mild lung disease at the outset of infection have remained clinically stable, whereas, only six of 28 patients with severe disease remained stable, three of whom were transiently infected with P . cepacia . The prevalence of P . cepacia at the time of death fluctuated between 12.5% and 26.9% during the study period.

FEMS Microbiol Lett, 1993 Apr 1, 108(2), 211 - 7
Degradation of Aroclor 1221 in soil by a hybrid pseudomonad; Havel J et al.; The hybrid Pseudomonas cepacia strain JHR22 was tested for its ability to degrade Aroclor 1221 in soil . The influence of supplements--mineral salts and trace elements--on the degradation was investigated . Disappearance of Aroclor 1221 congeners, occurrence of metabolites, and release of chloride were measured under different conditions . After 45 days the hybrid organism, strain JHR22, was still present at high numbers in soil, independently of whether the soil had been sterilized prior to inoculation or not . There was only a minor difference in degradation efficiency between sterilized and untreated soil with about 70% release of chloride when 10(7) cells/g soil were inoculated . The whole hybrid pathway, originating from three different strains, was found to be stable under the conditions tested . Mineral salts did not significantly affect the degradation rate or survival of the hybrid strain.

J Clin Microbiol, 1993 Apr, 31(4), 788 - 92
Marked phenotypic variability in Pseudomonas cepacia isolated from a patient with cystic fibrosis; Larsen GY et al.; Characterization of the epidemiology of Pseudomonas cepacia colonization in cystic fibrosis is difficult because of the phenotypic variability of isolates . A single sputum culture may yield colonies which differ in morphology, antibiotic susceptibility, and pigment production . We examined serial P . cepacia isolates from a cystic fibrosis patient which the clinical laboratory identified as separate strains; these were selected on the basis of isolation date and culture site . An attempt was made to sample at multiple time points and, at a single time point, from three different culture sites . Ribotype analysis, using both the standard Southern blot technique and a recently reported method which uses the polymerase chain reaction, was used to distinguish unique P . cepacia strains . Characterization included comparison of antibiotic susceptibility, plasmid content, and outer membrane protein (OMP) patterns . rRNA analysis demonstrated that all isolates had the same ribotype, consistent with their being derivatives of the same strain . Antibiotic susceptibility testing revealed variability among both same-date and same-site isolates . Screening for plasmid DNA identified three groups of isolates; both same-date and same-site isolates demonstrated variability . OMP profiles were similar, but at least six distinct patterns were identified . For the six same-date isolates, five different OMP patterns were identified . For the 10 same-site isolates from different dates, five of the six OMP patterns were represented . We have demonstrated marked phenotypic variability in 14 strains of P . cepacia isolated from different sites and at different times from a single colonized patient . Ribotyping identified all the isolates as derivatives of a single strain; thus, the diversity of phenotypes appears to be the result of differential gene expression.

Biochem Biophys Res Commun, 1993 Mar 31, 191(3), 1145 - 51
Expression of non-ADP-ribosylatable, diphtheria toxin-resistant elongation factor 2 in Saccharomyces cerevisiae; Kimata Y et al.; Eucaryotic elongation factor 2 (EF-2) contains a post-translationally modified histidine residue termed diphthamide that is specifically ADP-ribosylated by diphtheria toxin (DT) or Pseudomonas exotoxin A . To analyze the potential physiological role of ADP-ribosylation of EF-2 by cellular ADP-ribosyl transferase, we constructed DT-resistant, non-ADP-ribosylatable Saccharomyces cerevisiae EF-2 by site-directed mutagenesis and expressed the mutant EF-2 in yeast . Substitution of Arg for Gly(701) in yeast EF-2 conferred complete resistance to DT in vivo and in vitro . However, when only non-ribosylatable EF-2 was expressed in cells using genetic manipulation, the mutated EF-2 did not affect vegetative cell growth, mating, sporulation and germination of ascospores.

J Mol Biol, 1993 Mar 20, 230(2), 473 - 82
Structural studies of the enveloped dsRNA bacteriophage phi 6 of Pseudomonas syringae by Raman spectroscopy . II . Nucleocapsid structure and thermostability of the virion, nucleocapsid and polymerase complex; Bamford JK et al.; Structures and thermostabilities of the double-stranded (ds) RNA bacteriophage phi 6 and of its isolated nucleocapsid-polymerase complex (nucleocapsid core) and dsRNA components have been investigated by Raman spectroscopy . The spectra show that proteins of the phi 6 virion are collectively deficient in beta-sheet secondary structure . In particular, the major protein (P8) of the outer spherical shell of the phi 6 nucleocapsid exhibits a secondary structure dominated largely by alpha-helix and irregular conformations . The absence of appreciable beta-structure in the P8 subunit suggests a tertiary conformation lacking the beta-barrel motif common to subunits of most other spherical viral capsids . In addition, the Raman spectra show that subunits of the dodecahedral nucleocapsid core are also predominantly alpha-helical . The results thus indicate a largely alpha-helical secondary structure for the major subunit (P1) of the phi 6 nucleocapsid core, as well as for the P8 subunit of the outer spherical shell . Using Raman difference spectroscopy, we demonstrate that proteins of the nucleocapsid core (P1, P2, P4 and P7) interact extensively with the packaged phi 6 RNA genome, and further, that conformational stability of the packaged RNA is reduced upon removal from the core . Also, we find that proteins of the phi 6 nucleocapsid are significantly more thermostable than proteins of the viral membrane envelope, which are reported in the accompanying paper (Li et al., 1993) . The present results suggest that both the architectural principles and modes of protein-RNA interaction in the phi 6 virion differ fundamentally from those of icosahedral single-stranded RNA viruses . Both Raman and circular dichroism spectra indicate that the dsRNA genome of phi 6 is an A-form structure . The Raman marker bands signify the presence only of C3'-endo/anti nucleoside conformers . The Raman signature of dsRNA, revealed in the spectrum of the phi 6 genome, is discussed here as a model for assessing base-pairing and base-stacking interactions in other ribonucleoprotein assemblies.

J Mol Biol, 1993 Mar 20, 230(2), 461 - 72
Structural studies of the enveloped dsRNA bacteriophage phi 6 of Pseudomonas syringae by Raman spectroscopy . I . The virion and its membrane envelope; Li T et al.; We report and interpret the first Raman spectrum of a double-stranded RNA virus containing a membrane envelope . Spectra of the native bacteriophage phi 6 and of its isolated host-attachment (spike) protein and phospholipid-free core assembly were collected from aqueous solutions over a wide range of temperature . Comparison of the vibrational spectra by digital difference methods permits the following structural conclusions regarding molecular constituents of the fully assembled virion . (1) The double-stranded RNA, phospholipid and protein components of the phage exhibit Raman amplitudes in accordance with their biochemically determined compositions in the native virion (10, 20 and 70%, respectively) . (2) alpha-Helix and irregular conformations are the dominant secondary structures in proteins of both the viral membrane and nucleocapsid . This represents a departure from previously examined icosahedral phage and plant viruses, which are dominated by beta-sheet structures . (3) The phospholipids of the viral membrane are liquid crystalline throughout the determined range of virus thermostability (0 to 40 degrees C) . (4) The P3 spike protein of phi 6, which is anchored to, but not sequestered within the viral membrane, is largely alpha-helical (approximately 35%) and highly thermolabile . Denaturation of P3 at temperatures above 30 degrees C leads to appreciable loss (approximately 20%) of alpha-helix in favor of beta-strand structure, and alters significantly the environments of many aromatic side-chains . (5) The secondary structures of integral membrane proteins of phi 6 are overwhelmingly alpha-helical (approximately 70 to 80%) and also thermolabile . In contrast to P3, which exhibits aspartate and glutamate carboxyls in the ionized form (CO2-), the integral membrane proteins exhibit only protonated carboxyl groups (COOH) . Treatment of phi 6 with butylated hydroxytoluene (BHT), which has been shown to remove the P3 spike protein, does not significantly perturb phospholipids and associated integral proteins of the viral membrane or structural proteins and packaged double-stranded RNA of the nucleocapsid . However, P3 subunits, which are recovered after BHT treatment, exhibit radically altered secondary and tertiary structures, including the loss of most subunit alpha-helices . Among the P3 side-chains affected by BHT treatment, we note a general trend toward greater hydrophilicity and greater solvent exposure of the aromatic residues Trp and Tyr . On the other hand, the cysteine sulfhydryl groups of the BHT-isolated P3 monomer are not solvent exposed and function as strong hydrogen-bond donors in the protein core.(ABSTRACT TRUNCATED AT 400 WORDS)

FEMS Microbiol Lett, 1993 Mar 15, 108(1), 1 - 5
Phenylacetate-coenzyme A ligase is induced during growth on phenylacetic acid in different bacteria of several genera; Vitovski S; Nine different bacterial strains that utilise phenylacetic acid as the only carbon and energy source were isolated from samples of different geographical origin . The isolates were characterised taxonomically and physiologically . Evidence is presented that in all the isolates as well as in four previously isolated control strains with the ability to utilize phenylacetic acid, the enzyme phenylacetate-CoA ligase is specifically induced during growth on phenylacetic acid . The Michaelis constant (Km) in one Pseudomonas strain was sufficiently low (-1 mM) to suggest that the enzyme may have a role in phenylacetic acid metabolism.

J Biol Chem, 1993 Mar 5, 268(7), 5302 - 8
Characterization of single-chain antibody (sFv)-toxin fusion proteins produced in vitro in rabbit reticulocyte lysate; Nicholls PJ et al.; Chimeric proteins consisting of a fusion between binding-deficient mutants of diphtheria toxin (DT) or Pseudomonas exotoxin A (PE) and a single-chain antibody (E6 sFv) against the human transferrin receptor (TfnR) were expressed in a rabbit reticulocyte lysate system . Molecules utilizing PE40 (the carboxyl terminus 40 kDa of PE, lacking the binding domain) exhibited significant E6 sFv-mediated, cell type-specific cytotoxicity (IC50 1 x 10(-10) M) against a human erythroleukemia-derived cell line, K562 . In contrast, a fusion protein between the same sFv and a DT mutant, DTM1 (containing two amino acid substitutions in the binding domain {S(508)F, S(525)F}) was not significantly cytotoxic, despite being enzymatically active . A tripartite protein in the form NH2-DTM1-E6 sFv-PE40-COOH exhibited cytotoxicity comparable to that of the PE40-sFv fusion (IC50 1 x 10(-10) M), suggesting that the deficit in activity of DTM1-sFv is not a function of misfolding of the sFv moiety or of a reduced ability to bind TfnR . In contrast to DTM1-E6 sFv, a fusion protein between a second DT mutant, CRM 107 {S(525)F}, and the E6 sFv was specifically cytotoxic (IC50 1 x 10(-9) M), and toxicity could be blocked by addition of excess E6 antibody . The cell-free in vitro expression system we describe is rapid and may be used to express functional toxin-sFv fusion proteins . No protein refolding procedures are required, and the technique may be used to express proteins which, due to restrictions imposed on manipulation of toxin-encoding genes in Escherichia coli, could not be produced by more conventional methods.

J Biol Chem, 1993 Mar 5, 268(7), 4853 - 62
Heparin-binding transforming growth factor alpha-Pseudomonas exotoxin A . A heparan sulfate-modulated recombinant toxin cytotoxic to cancer cells and proliferating smooth muscle cells; Mesri EA et al.; TGF alpha-PE40, a recombinant toxin in which transforming growth factor alpha (TGF alpha) is fused to a mutant form of Pseudomonas exotoxin, is selectively cytotoxic to cells bearing epidermal growth factor (EGF) receptors . Heparin binding EGF-like growth factor is a potent mitogen for smooth muscle cells capable of binding to both the EGF receptor and to immobilized heparin (Higashiyama, S., Abraham, J., Miller, J., Fiddes, J., and Klagsbrun, M . (1991) Science 251, 936-938) . To study the effect of the heparin-binding domain in a chimeric toxin targeted to the EGF receptor, we fused the DNA sequence corresponding to the putative NH2-terminal heparin-binding (HB) domain of HB-EGF to chimeric toxins composed of TGF alpha and two different recombinant forms of Pseudomonas exotoxin (PE) . One of these is a truncated form of PE devoid of the binding domain (TGF alpha-PE38); another is a mutant form of full-length toxin containing inactivating mutations in the binding domain and an altered carboxyl terminus (TGF alpha-PE4EKDEL) . The resulting chimeric toxins HB-TGF alpha-PE38 and HB-TGF alpha-PE4EKDEL were expressed in Escherichia coli as inclusion bodies, refolded, and purified by heparin affinity chromatography . Both of the toxins were eluted from heparin at 0.8 M NaCl, in contrast to their respective TGF alpha toxins which were eluted at 0.15 M . Binding studies on A431 cells showed that the HB-TGF alpha toxins bound to the EGF receptor with an affinity similar to that of the TGF alpha toxins . However, cell killing studies on a panel of malignant cell lines showed that cytotoxicity was strongly affected by the presence of the HB domain . Cell lines expressing high numbers of EGF receptors such as A431 and KB were less sensitive to toxins containing the HB domain . Cells with low number of EGF receptors had similar responses to both types of toxins (MCF-7 and LNCaP) or were more sensitive to the toxin with the added HB domain (HEP-G2) . HB-TGF alpha-PE4EKDEL was over 10-fold more cytotoxic against proliferating vascular smooth muscle cells (VSMC) than to quiescent VSMC . Moreover, HB-TGF alpha-PE4EKDEL was 6-fold more potent than TGF alpha-PE4EKDEL to proliferating VSMC . Competition studies with EGF and/or heparin showed that heparin blocks the cytotoxicity of HB-TGF toxins and the inhibitory action of heparin is stronger in cells expressing lower number of EGF receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochim Biophys Acta, 1993 Mar 5, 1162(1-2), 89 - 92
Amino-acid sequence of cytochrome c-553 from Desulfovibrio desulfuricans Norway; Bruschi M et al.; The amino-acid sequence of the cytochrome c-553 from Desulfovibrio desulfuricans Norway has been determined and compared with that of two different cytochromes c-553 from D . vulgaris already described and with that cytochrome c-551 from Pseudomonas . This low-molecular-weight monohemic cytochrome comprises 80 amino acids and has the typical characteristics of small cytochromes such as mitochondrial cytochromes . Secondary-structure predictions are deduced from sequence data and are compared with X-ray three-dimensional structures of low-molecular-weight cytochrome c . The phylogenetic situation of Desulfovibrio cytochromes c-533 in the cytochrome c superfamily is discussed.

Cornea, 1993 Mar, 12(2), 138 - 41
Ciprofloxacin ointment versus ciprofloxacin drops for therapy of experimental Pseudomonas keratitis; Hobden JA et al.; To compare the efficacy of ciprofloxacin hydrochloride ointment with that of ciprofloxacin drops for treatment of experimental Pseudomonas keratitis, rabbit eyes were infected by intrastromal injection of 10(3) colony-forming units (CFU) log-phase P . aeruginosa . Infected and uninfected eyes were treated with either 0.3% ciprofloxacin ointment applied hourly for 6 h or 0.3% ciprofloxacin drops applied every 15 min for 6 h . Infected eyes treated with the ointment or drop vehicle alone served as placebo controls . Ciprofloxacin ointment significantly reduced the number of viable bacteria (CFU) per cornea more than four logs compared to the placebo control (p < 0.0001) . Ciprofloxacin drops significantly reduced the number of bacteria (CFU) per cornea > 7 logs as compared with placebo-treated controls (p < 0.0001) . Ciprofloxacin ointment may be a useful adjunct to conventional topical drops for therapy of bacterial keratitis.

Appl Environ Microbiol, 1993 Mar, 59(3), 860 - 6
Selection of Pseudomonas sp . strain HBP1 Prp for metabolism of 2-propylphenol and elucidation of the degradative pathway; Kohler HP et al.; A mutant of Pseudomonas sp . strain HBP1, originally isolated on 2-hydroxybiphenyl, was selected for the ability to grow on 2-propylphenol as the sole carbon and energy source . In the mutant strain, which was designated as Pseudomonas sp . strain HBP1 Prp, the cellular induction mechanism involved in the synthesis of the NADH-dependent monooxygenase is changed . 2-Propylphenol, which is known to be a substrate of the monooxygenase, does not induce formation of the monooxygenase in the wild type but does have an induction effect in the mutant strain . Furthermore, in contrast to the wild type, mutant strain HBP1 Prp constitutively produces a small amount of monooxygenase and metapyrocatechase . The enzymes from strain HBP1 Prp catalyzing the first three steps in the degradation of 2-propylphenol--the NADH-dependent monooxygenase, the metapyrocatechase, and the meta fission product hydrolase--were partially purified, and their activities were measured . The product of the monooxygenase activity was identified by mass spectrometry as 3-propylcatechol . The metapyrocatechase used this compound as a substrate and produced a yellow meta fission product that was identified by mass spectrometry as 2-hydroxy-6-oxo-nona-2,4- dienoate . Butyrate could be detected as a product of the meta fission product hydrolase in crude cell extract of 2-propylphenol-grown cells, as well as an intermediate in culture broths during growth on 2-propylphenol . All three enzymes expressed highest activities for the metabolites of the degradation of 2-hydroxybiphenyl.

FEMS Microbiol Lett, 1993 Mar 1, 107(2-3), 151 - 5
A natural isolate of Pseudomonas maltophila which degrades aromatic sulfonic acids; Lee NA et al.; A natural isolate, designated BSA56, which was originally selected for growth with benzene sulfonic acid as sole carbon and energy source, was identified as a strain of Pseudomonas maltophila . Strain BSA56 grew on a wide range of aromatic sulfonic acids and was shown to release sulfite from benzene sulfonic acid and 2-naphthalene sulfonic acid . Although it also grew on toluene sulfonic acid and pyridine sulfonic acid, no significant sulfite release was observed with these substrates . Release of sulfite from benzene sulfonic acid was greatly promoted by the presence of glycerol . The ability to release sulfite was induced by growth in the presence of benzene sulfonic acid and was repressed almost entirely by substrates allowing rapid growth such as acetate . Strain BSA56 grew better at 30 degrees C than 37 degrees C on most aromatic substrates, but the reverse was true for most aromatic sulfonates . Several mutants of BSA56 were isolated with defects in benzoate, salicylate, or gentisate metabolism . However, all these mutants retained the ability to degrade the aromatic sulfonates.

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