Plant Cell Physiol, 2000 Apr, 41(4), 458 - 64 Isolation of the gene for EILP, an elicitor-inducible LRR receptor-like protein, from tobacco by differential display; Takemoto D et al.; We screened tobacco genes, which are differentially expressed in response to a fungal elicitor, and have isolated a gene which codes for a leucine-rich repeat (LRR) protein closely related to Cf genes in tomato . The EILP (elicitor inducible LRR protein) gene encodes 95 kDa protein, which consists of a putative membrane spanning region, 28 leucine-rich repeats and some N-linked glycosylation sites, and shows high homology to Cf-2/Cf-5 family genes . Southern blot analysis revealed the presence of some genes homologous to EILP in tobacco, like Cf genes in tomato . The expression of EILP was low at the basal level and increased by treatment with elicitor, implying that EILP is involved in both preexisting and inducible surveillance systems . The expression of EILP was activated by a non-pathogen, Pseudomonas syringae pv . glycinea, and in a delayed fashion by the tobacco pathogen P . syringae pv . tabaci, suggesting that the product of EILP may be involved in non-host disease resistance in tobacco. J Bacteriol, 2000 Jul, 182(13), 3784 - 93 Genetic investigation of the catabolic pathway for degradation of abietane diterpenoids by Pseudomonas abietaniphila BKME-9; Martin VJ et al.; We have cloned and sequenced the dit gene cluster encoding enzymes of the catabolic pathway for abietane diterpenoid degradation by Pseudomonas abietaniphila BKME-9 . The dit gene cluster is located on a 16.7-kb DNA fragment containing 13 complete open reading frames (ORFs) and 1 partial ORF . The genes ditA1A2A3 encode the alpha and beta subunits and the ferredoxin of the dioxygenase which hydroxylates 7-oxodehydroabietic acid to 7-oxo-11,12-dihydroxy-8, 13-abietadien acid . The dioxygenase mutant strain BKME-941 (ditA1::Tn5) did not grow on nonaromatic abietanes, and transformed palustric and abietic acids to 7-oxodehydroabietic acid in cell suspension assays . Thus, nonaromatic abietanes are aromatized prior to further degradation . Catechol 2,3-dioxygenase activity of xylE transcriptional fusion strains showed induction of ditA1 and ditA3 by abietic, dehydroabietic, and 7-oxodehydroabietic acids, which support the growth of strain BKME-9, as well as by isopimaric and 12, 14-dichlorodehydroabietic acids, which are diterpenoids that do not support the growth of strain BKME-9 . In addition to the aromatic-ring-hydroxylating dioxygenase genes, the dit cluster includes ditC, encoding an extradiol ring cleavage dioxygenase, and ditR, encoding an IclR-type transcriptional regulator . Although ditR is not strictly required for the growth of strain BKME-9 on abietanes, a ditR::Km(r) mutation in a ditA3::xylE reporter strain demonstrated that it encodes an inducer-dependent transcriptional activator of ditA3 . An ORF with sequence similarity to genes encoding permeases (ditE) is linked with genes involved in abietane degradation. Cancer Res, 2000 Jun 1, 60(11), 2981 - 7 Structure, function, and targeting of interleukin 4 receptors on human head and neck cancer cells; Kawakami K et al.; Despite advances in diagnosis and treatment, survival rates for patients with head and neck cancer have remained unchanged for the last 30 years . In an attempt to develop novel therapeutic agents, we have observed that a variety of murine and human carcinoma cells expresses high levels of receptors for interleukin 4 (IL-4) in vitro and in vivo . Here, we demonstrate that 17 head and neck cancer cell lines also express surface IL-4 receptors (IL-4R) and IL-4 binds to IL-4R on one cell line studied with low affinity ((k)d = 37.9 +/- 0.4 nM) . The investigation of the subunit structure of IL-4R demonstrated that head and neck cancer cell lines expressed mRNA for IL-4R beta chain (also known as IL-4R alpha) and IL-13R alpha' chain (also known as IL-13R alpha1) . However, no cell line expressed IL-2R common gamma-chain, which is known to be shared with IL-4R in immune cells . IL-4R is functional because IL-4 strongly induced activation of signal transducers and activators of transcription 6 (STAT-6) in these cell lines . A fusion protein, IL4(38-37)-PE38KDEL, containing translocation and enzymatic domains of Pseudomonas exotoxin and a circularly permuted human IL-4 was found to be highly and specifically cytotoxic to IL-4R-positive head and neck cancer cells, as determined by protein synthesis inhibition assay and confirmed by clonogenic assay . IL4(38-37)-PE38KDEL induced DNA fragmentation and condensation of nuclei indicative of apoptotic cell death . These results establish uniform expression of IL-4R on head and neck cancer cell lines and IL-4 toxin IL4(38-37)-PE38KDEL as a novel therapeutic agent for the possible treatment of human head and neck cancers. Appl Biochem Biotechnol, 2000 Spring, 84-86, 971 - 80 Effect of C:N molar ratio on monomer composition of polyhydroxyalkanoates produced by Pseudomonas mendocina 0806 and Pseudomonas pseudoalkaligenus YS1; Hong K et al.; Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by bacteria . In this study, the effect of C:N molar ratio on the monomer composition of PHAs was investigated, including medium chain length PHA produced by Pseudomonas mendocina 0806 and PHA blends consisting of monomers of 3-hydroxybutyrate and medium chain length hydroxyalkanoate produced by Pseudomonas pseudoalkaligenus YS1 . It was observed that there were some fixed ranges of C:N molar ratio that affect the monomer composition of PHA independently of the substrate . For strain 0806, the ranges were C:N < 20, 20 < C:N < 200, and C:N > 200 . The monomer composition was constant among these ranges when using glucose and octanoate as the sole substrate . For strain YS1, the ranges were C:N < 20, 20 < C:N < 45, and C:N > 45 . These results are useful for controlling monomer composition in PHA production. Plant J, 2000 May, 22(4), 345 - 54 A resistance gene product of the nucleotide binding site -- leucine rich repeats class can form a complex with bacterial avirulence proteins in vivo; Leister RT et al.; Resistance (R) genes in plants mediate gene-for-gene disease resistance . The ligand-receptor model, which explains the gene-for-gene specificity, predicts a physical interaction between an elicitor, which is directly or indirectly encoded by an avirulence (avr) gene in the pathogen, and the corresponding R gene product . The nucleotide binding site (NBS) - leucine rich repeats (LRR) class of R genes is the largest known class of R genes . Here we report that an NBS-LRR R protein and its cognate Avr protein form a complex together in the plant cell . The Arabidopsis thaliana R genes RPS2 and RPM1 confer gene-for-gene disease resistance to strains of the phytopathogenic bacterium Pseudomonas syringae carrying the avr genes avrRpt2 and avrB, respectively . Using transient expression of these genes in Arabidopsis leaf mesophyll protoplasts, we first demonstrated that the protoplast system is appropriate for the investigation of the gene-for-gene recognition mechanism . Formation of an in vivo complex containing the RPS2 and AvrRpt2 proteins was demonstrated by co-immunoprecipitation of the proteins following expression of the genes in protoplasts . This complex contained at least one additional plant protein of approximately 75 kDa . Unexpectedly, RPS2 also formed a complex with AvrB . We speculate that complex formation between AvrRpt2 and RPS2 is productive and leads to the elicitation of the resistance response, whilst complex formation between AvrB and RPS2 is unproductive and possibly competes with complex formation between AvrRpt2 and RPS2. J Laryngol Otol, 2000 Apr, 114(4), 260 - 3 A prospective study of nasal disease in adult cystic fibrosis; Raj P et al.; Twenty-six adult cystic fibrosis patients were studied to compare nasal disease with their laboratory correlates including skin testing, immunoglobulin and Aspergillus fumigatus precipitin levels, saccharin testing and sputum cultures . Six patients were asymptomatic and all of these had negative skin tests, normal IgE levels and negative Aspergillus fumigatus precipitins . Thirteen patients had rhinitis, 12 had positive skin-testing for common allergens, 10 elevated IgE levels and nine positive Aspergillus fumigatus precipitins . Seven patients had polyps, all had normal IgE levels and negative Aspergillus fumigatus precipitins, six had positive skin testing for common allergens . There also appeared to be a relationship between Pseudomonas spp . colonization and positive skin testing. Adv Drug Deliv Rev, 1998 Apr 6, 31(1-2), 53 - 88 Immunotoxins for targeted cancer therapy; Pastan I I et al.; Immunotoxins constitute a new modality for the treatment of cancer, since they target cells displaying specific surface-receptors or antigens . Immunotoxins contain a ligand such as a growth factor, monoclonal antibody, or fragment of an antibody which is connected to a protein toxin . After the ligand subunit binds to the surface of the target cell, the molecule internalizes and the toxin kills the cell . Bacterial toxins which have been targeted to cancer cells include Pseudomonas exotoxin and diphtheria toxin, which are well suited to forming recombinant single-chain or double-chain fusion toxins . Plant toxins include ricin, abrin, pokeweed antiviral protein, saporin and gelonin, and have generally been connected to ligands by disulfide-bond chemistry . Immunotoxins have been produced to target hematologic malignancies and solid tumors via a wide variety of growth factor receptors and antigens . Challenges facing the clinical application of immunotoxins are discussed. Arch Pharm Res, 2000 Apr, 23(2), 187 - 95 Characterization of the pcbE gene encoding 2-hydroxypenta-2,4-dienoate hydratase in Pseudomonas sp . DJ-12; Lim JC et al.; Nucleotide sequence extending 2,3-dihydroxybiphenyl 1,2-dioxygenase gene (pcbC) and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase gene (pcbD) of Pseudomonas sp . DJ-12 was previously analyzed and the two genes were present in the order of pcbD-pcbC preceded by a promoter from Pseudomonas sp . DJ-12 . In this study, a 3.8-kb nucleotide sequence located downstream of the pcbC gene was analyzed to have three open reading frames (ORFs) that are designated as orf1, pcbE and orf2 genes . All of the ORFs were preceded by each ribosome-binding sequence of 5-GGAXA-3 (X=G or A) . However, no promoter-like sequence and transcription terminator sequence were found in the analyzed region, downstream of pcbC gene . Therefore, the gene cluster appeared to be present in the order of pcbD-pcbC-orf1-pcbE-orf2 as an operon, which is unique organization characterized so far in biphenyl- and PCB-degrading bacteria . The orf1 gene was composed of 1,224 base pairs which can encode a polypeptide of molecular weight 44,950 containing 405 amino acid residues . A deduced amino acid sequence of the orf1 gene product exhibited 21-33% identity with those of indole dioxygenase and phenol hydroxylase components . The pcbE gene was composed of 783 base pairs encoding 2-hydroxypenta-2,4-dienoate hydratase involved in the 4-chlorobiphenyl catabolism . The orf2 gene was composed of 1,017 base pairs encoding a polypeptide of molecular weight 37,378 containing 338 amino acid residues . A deduced amino acid sequence of the orf2 gene product exhibited 31% identity with that of a nitrilotriacetate monooxygenase component. Biotechnol Prog, 2000 May-Jun, 16(3), 372 - 7 Comparative fatty acid selectivity of lipases in esterification reactions with glycerol and diol analogues in organic media; Lee CH et al.; Reaction selectivity of Pseudomonas cepacia, Rhizomucor miehei, and Candida antarctica B lipases was assessed in multicompetitive esterification reaction mixtures containing an homologous series of n-chain even carbon number fatty acid (FA; C4-C18) substrates and a single alcohol cosubstrate (glycerol, 1,2-propanediol (1,2-PD), or 1, 3-propanediol (1,3-PD)) in tert-butyl methyl ether at water activity of 0.69 or 0.90 and a reaction temperature of 35 degrees C . For P . cepacia lipase, the ordinal patterns of FA selectivities observed were, with glycerol, C8 > C10, C6, C16 > other FA; with 1,2-PD and 1, 3-PD, C16 > C8 > C14 > other FA . For R . miehei lipase, the ordinal patterns of FA selectivities observed were, with glycerol, C8 > C12 > C10, C14 > other FA; with 1,2-PD and 1,3-PD, C8 > C12 > other FA . For C . antarctica B lipase, the ordinal patterns of FA selectivities observed were, with glycerol, C8 > C10, C6, C12 > other FA; with 1, 2-PD, C8 > C10, C6 > other FA; and with 1,3-PD, C8 > C10 > C6 > other FA . The differences in selectivity among FA ranged up to 16-fold, depending upon the lipase and alcohol cosubstrate used . These findings represent intrinsic and substrate-modulated features of FA selectivities that are of particular relevance to the use of lipases for acylglycerol synthesis reactions. Appl Environ Microbiol, 2000 Jun, 66(6), 2673 - 7 Detection of Pseudomonas savastanoi pv . savastanoi in olive plants by enrichment and PCR; Penyalver R et al.; The sequence of the gene iaaL of Pseudomonas savastanoi EW2009 was used to design primers for PCR amplification . The iaaL-derived primers directed the amplification of a 454-bp fragment from genomic DNA isolated from 70 strains of P . savastanoi, whereas genomic DNA from 93 non-P . savastanoi isolates did not yield this amplified product . A previous bacterial enrichment in the semiselective liquid medium PVF-1 improved the PCR sensitivity level, allowing detection of 10 to 100 CFU/ml of plant extract . P . savastanoi was detected by the developed enrichment-PCR method in knots from different varieties of inoculated and naturally infected olive trees . Moreover, P . savastanoi was detected in symptomless stem tissues from naturally infected olive plants . This enrichment-PCR method is more sensitive and less cumbersome than the conventional isolation methods for detection of P . savastanoi. Mol Plant Microbe Interact, 2000 Jun, 13(6), 592 - 8 A cluster of mutations disrupt the avirulence but not the virulence function of AvrPto; Shan L et al.; avrPto in Pseudomonas syringae pv . tomato encodes an avirulence protein that triggers race-specific resistance in tomato plants carrying Pto . The AvrPto protein is secreted from P . syringae pv . tomato to plant cells through the type III secretion pathway and activates race-specific resistance by a direct interaction with the Pto protein . Here we report that avrPto enhances the virulence of P . syringae pv . tomato in a strain-dependent manner in tomato plants lacking Pto . To determine whether the virulence function can be structurally separated from the avirulence function, we examined the virulence activity of a group of AvrPto mutants that carry single amino acid substitutions and lack the avirulence activity on tomato plants . Three mutants that were clustered in the center of AvrPto exhibited virulence activity in tomato plants with or without Pto . The rest of the mutations abolished the virulence . The identification of these mutants suggested that the avirulence function of AvrPto can be structurally separated from the virulence function. Indian J Pediatr, 1996 Mar-Apr, 63(2), 189 - 98 Cystic fibrosis--an Indian perspective on recent advances in diagnosis and management; Kabra SK et al.; Cystic fibrosis (CF) is a common inherited disorder in caucasians . The estimated incidence of CF in Asians varies from 1:10,000 to 1:12,000 . Indian data is restricted to few case reports . The gene for CF is located on the long arm of chromosome 7 at position 7q13 . There are more than 300 identified mutations in CF . The basic defect in CF is a mutational change in the gene for chloride conductance channel . Failure of chloride conductance by epithelial cells leads to dehydration of secretions that are too viscid and difficult to clear . The disease is characterized by abnormal secretions in the respiratory, gastrointestinal and reproductive tract and sweat glands . The common clinical manifestations include meconium ileus in neonatal period, recurrent lower respiratory tract infections (pseudomonas pneumonia, bronchiectasis), steatorrhoea, azoospermia, and in late stages hepatobiliary and endocrine pancreatic dysfunctions . The diagnosis of disease is established by clinical criteria and sweat chloride concentration more than 60 mEq/L . Facilities for DNA diagnosis of common CF mutations are now available in India . The treatment of CF includes early diagnosis, daily clearance of respiratory passages, appropriate antibiotic therapy, aerosolised recombinant human DNase and antibiotics, and nutritional supplementation . The latter include changes in diet composition, pancreatic enzyme supplementation and vitamins and trace mineral supplementation . Gene therapy for the pulmonary manifestations is being tried in a number of centres abroad . Other considerations include heart lung transplantation and ameloride inhalation therapy. Blood, 2000 Jun 1, 95(11), 3506 - 13 Interleukin-13 fusion cytotoxin as a potent targeted agent for AIDS-Kaposi's sarcoma xenograft; Husain SR et al.; Clinically advanced and rapidly progressive AIDS-associated Kaposi sarcoma (AIDS-KS) tumors require an aggressive tumor-directed therapy . We have observed that AIDS-KS cells express high levels of receptors for immune regulatory cytokine, interleukin-13 (IL-13) . Two tumorigenic AIDS-KS cell lines, KS Y-1 and KS-imm, expressed 4560 and 9480 IL-13 binding sites per cell with an affinity (kd) of approximately 0.9 and 3.7 nmol/L, respectively . IL-13 cytotoxin IL13-PE38QQR, consisting of human IL-13 and a derivative of Pseudomonas exotoxin, is specifically cytotoxic to KS tumor cells . Systemic and loco regional administration of IL13-PE38QQR in immunodeficient mice with established human KS tumors produced remarkable antitumor activity . Three intratumoral (IT) injections of IL-13 toxin (250 microg/kg per dose) on alternate days (qod) or 5 daily (qd) IT injections with lower doses (50 or 100 microg/kg per dose) resulted in a complete regression of established subcutaneous tumors in most animals . Daily IT treatment with 250 microg/kg of IL-13 toxin in another KS-derived cell line also produced complete responses . Twice daily intraperitoneal injections of IL13-PE38QQR (25 or 50 microg/kg per dose) for 10 days (total injections = 20) also completely eradicated KS Y-1 tumors . Intravenous administration of IL13-PE38QQR also suppressed tumor growth; however, complete responses were not observed . All animals tolerated the therapeutic doses of IL-13 toxin without any visible signs of toxicity . The efficacy of receptor-directed IL13-PE38QQR therapy in mice warrants further exploration of this drug for AIDS-KS treatment. Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 9 - 18 Pseudomonas brassicacearum sp . nov . and Pseudomonas thivervalensis sp . nov., two root-associated bacteria isolated from Brassica napus and Arabidopsis thaliana; Achouak W et al.; Bacteria isolates phenotypically related to Pseudomonas corrugata have frequently been isolated from the rhizosphere of Arabidopsis thaliana and Brassica napus grown on different soils . 16S rDNA (rrs) gene sequencing, DNA-DNA hybridization, biochemical characterization and siderophore typing showed that these isolates belong to two different species that are distinct from other species of the genus Pseudomonas, including P . corrugata . A description of properties of these two new species is given based on the study of 16 isolates . Proposed names are Pseudomonas brassicacearum (10 strains studied) and Pseudomonas thivervalensis (6 strains studied) . The type strain of Pseudomonas brassicacearum is CFBP 11706T and that of Pseudomonas thivervalensis is CFBP 11261T. Biochim Biophys Acta, 2000 May 23, 1478(2), 201 - 10 Overproduction in Escherichia coli, purification and characterization of a family I.3 lipase from Pseudomonas sp . MIS38; Amada K et al.; Determination of the nucleotide sequence of the gene encoding a lipase from Pseudomonas sp . MIS38 (PML) revealed that PML is a member of the lipase family I.3 and is composed of 617 amino acid residues with a calculated molecular weight of 64510 . Recombinant PML (rPML) was overproduced in Escherichia coli in an insoluble form, solubilized in the presence of 8 M urea, purified in a urea-denatured form and refolded by removing urea in the presence of the Ca(2+) ion . Gel filtration chromatography suggests that this refolded protein is monomeric . rPML showed relatively broad substrate specificities and hydrolyzed glyceryl tributyrate and olive oil with comparable efficiencies . rPML was active only in the form of a holo-enzyme, in which at least 12 Ca(2+) ions bound . These Ca(2+) ions bound too tightly to be removed from the protein upon dialysis, but were removed from it upon EDTA treatment . The resultant apo-enzyme was fully active in the presence of 10 mM CaCl(2), but was inactive in the absence of the Ca(2+) ion . PML has a GXSXG motif, which is conserved in lipases/esterases and generally contains the active-site serine . The mutation of Ser(207) within this motif to Ala completely inactivated PML, suggesting that Ser(207) is the active-site serine of PML. Genetika, 2000 Apr, 36(4), 459 - 69 {Molecular genetic analysis of the Tn5041 transposition system}; Kholodii GIa et al.; A study was made of the transposition of the mercury resistance transposon Tn5041 which, together with the closely related toluene degradation transposon Tn4651, forms a separate group in the Tn3 family . Transposition of Tn5041 was host-dependent: the element transposed in its original host Pseudomonas sp . KHP41 but not in P . aeruginosa PAO-R and Escherichia coli K12 . Transposition of Tn5041 in these strains proved to be complemented by the transposase gene (tnpA) of Tn4651 . The gene region determining the host dependence of Tn5041 transposition was localized with the use of a series of hybrid (Tn5041 x Tn4651) tnpA genes . Its location in the 5'-terminal one-third of the transposase gene is consistent with the data that this region is involved in the formation of the transposition complex in transposons of the Tn3 family . As in other transposons of this family, transposition of Tn5041 occurred via cointegrate formation, suggesting its replicative mechanism . However, neither of the putative resolution proteins encoded by Tn5041 resolved the cointegrates formed during transposition or an artificial cointegrate in E . coli K12 . Similar data were obtained with the mercury resistance transposons isolated from environmental Pseudomonas strains and closely related to Tn5041 (Tn5041 subgroup). Biotechnol Bioeng, 2000 Jul 5, 69(1), 107 - 12 Transformation of mono- and dichlorinated phenoxybenzoates by phenoxybenzoate-dioxygenase in pseudomonas pseudoalcaligenes POB310 and a modified diarylether-metabolizing bacterium; Halden RU et al.; Pseudomonas pseudoalcaligenes POB310 contains genes that encode phenoxybenzoate dioxygenase . The enzyme transforms mono- and dichlorinated phenoxybenzoates to yield protocatechuate that is used as a growth substrate and chlorophenols that are nonmetabolizable . Mass spectral analysis of (18)O metabolites obtained from the protocatechuate 3,4-dioxygenase-deficient mutant, POB310-B1, suggested that the reaction mechanism is a regioselective angular dioxygenation . A cloning vector containing reaction relevant genes (pD30.9) was transferred into Pseudomonas sp . strain B13 containing a modified ortho-cleavage pathway for aromatic compounds . The resultant Pseudomonas sp . strain B13-D5 (pD30.9) completely metabolized 3-(4-chlorophenoxy)benzoate . During growth on 3-phenoxybenzoate, strain B13-D5 (pD30.9) (K(s) = 0.70+/-0.04 mM, mu(max) = 0.45+/-0.03 h(-1), t(d) = 1.5 h, Y = 0.45+/-0.03 g bio- mass x g substrate(-1)) was better adapted to low substrate concentrations, had a faster rate of growth, and a greater yield than POB310 (K(s) = 1.13+/-0.06 mM, mu(max) = 0.31+/-0.02 h(-1), t(d) = 2.2 h, Y = 0.39+/-0.02 g biomass . g substrate(-1)) . Biotechnol Bioeng, 2000 Jul 5, 69(1), 91 - 100 Production of enantiopure styrene oxide by recombinant Escherichia coli synthesizing a two-component styrene monooxygenase; Panke S et al.; A whole cell biocatalytic process was developed to enable the efficient oxidation of styrene to chiral (S)-styrene oxide with an enantiomeric excess better than 99% . Recombinant Escherichia coli cells were employed to express the genes styAB encoding the styrene monooxygenase of Pseudomonas sp . strain VLB120 from an expression plasmid utilizing the alk regulatory system of P . oleovorans GPo1 . The strains reached specific activities of up to 70 U* (g cell dry weight)(-1) in shake-flask experiments with glucose as the carbon source . An efficient two-liquid phase fed-batch process was established for the production of (S)-styrene oxide with hexadecane as an apolar carrier solvent and a nutrient feed consisting of glucose, magnesium sulfate, and yeast extract . Engineering of the phase fraction and the composition of organic phase and feed led to a 2-L scale process with maximal volumetric productivities of 2.2 g (S)-styrene oxide per liter liquid volume per hour . This optimized process was based completely on defined medium and used bis(2-ethylhexyl)phthalate as the apolar carrier solvent, which together with substrate and inducer consisted of 50% of the total liquid volume . Using this system, we were able to produce per liter liquid volume 11 g of enantiopure (S)-styrene oxide in 10 h . J Biotechnol, 2000 Apr 14, 79(1), 13 - 26 D-malate production by permeabilized Pseudomonas pseudoalcaligenes; optimization of conversion and biocatalyst productivity; Michielsen MJ et al.; For the development of a continuous process for the production of solid D-malate from a Ca-maleate suspension by permeabilized Pseudomonas pseudoalcaligenes, it is important to understand the effect of appropriate process parameters on the stability and activity of the biocatalyst . Previously, we quantified the effect of product (D-malate2 -) concentration on both the first-order biocatalyst inactivation rate and on the biocatalytic conversion rate . The effects of the remaining process parameters (ionic strength, and substrate and Ca2 + concentration) on biocatalyst activity are reported here . At (common) ionic strengths below 2 M, biocatalyst activity was unaffected . At high substrate concentrations, inhibition occurred . Ca2+ concentration did not affect biocatalyst activity . The kinetic parameters (both for conversion and inactivation) were determined as a function of temperature by fitting the complete kinetic model, featuring substrate inhibition, competitive product inhibition and first-order irreversible biocatalyst inactivation, at different temperatures simultaneously through three extended data sets of substrate concentration versus time . Temperature affected both the conversion and inactivation parameters . The final model was used to calculate the substrate and biocatalyst costs per mmol of product in a continuous system with biocatalyst replenishment and biocatalyst recycling . Despite the effect of temperature on each kinetic parameter separately, the overall effect of temperature on the costs was found to be negligible (between 293 and 308 K) . Within pertinent ranges, the sum of the substrate and biocatalyst costs per mmol of product was calculated to decrease with the influent substrate concentration and the residence time . The sum of the costs showed a minimum as a function of the influent biocatalyst concentration. Z Naturforsch {C}, 2000 Mar-Apr, 55(3-4), 146 - 52 Succinopyoverdins--a new variety of the pyoverdin chromophore; Lenz C et al.; Pseudomonas spp . of the fluorescent group produce siderophores (so-called pyoverdins) consisting of a peptide chain attached to a pyrimidoquinoline ring system which is derived from a condensation product of L-Dab and D-Tyr . Commonly several related compounds are found to accompany the pyoverdins having the same peptide chain, but differing in the heterocyclic part . The structure elucidation of a new variety (succinopyoverdin) is described here. Biometals, 1999 Dec, 12(4), 323 - 9 Structure of the pyoverdin PVD 2908--a new pyoverdin from Pseudomonas sp . 2908; Vossen W et al.; An unknown siderophore (pyoverdin) was isolated from the strain Pseudomonas sp . 2908 . The structure of the pyoverdin--called PVD 2908--was elucidated by spectroscopic methods and degradation studies . Some other siderophores were identified by LC/ESI-MS-screening based on the knowledge of PVD 2908. Anal Chem, 2000 May 1, 72(9), 2055 - 8 Immobilized parathion hydrolase: an amperometric sensor for parathion; Sacks V et al.; An amperometric enzyme biosensor for the direct measurement of parathion was developed . The biosensor is based on parathion hydrolase from Pseudomonas sp . isolated from contaminated soil . The enzyme, which was immobilized on a carbon electrode, catalyzes the hydrolysis of parathion to form p-nitrophenol, which was detected by its anodic oxidation . The enzymatic and electrochemical reactions were examined and optimized . Screen-printed electrodes and a microflow injection system provide the means to significantly reduce the volume of the detected samples . Pulsed techniques further increased the sensitivity of the measurement . The current signal was linearly related to the parathion concentration, and the detection limit was less than 1 ng/mL . The biosensor is rapid as well and can be used outdoors and indoors by a nonqualified person. AJNR Am J Neuroradiol, 2000 May, 21(5), 828 - 31 Imaging of mucormycosis skull base osteomyelitis; Chan LL et al.; Skull base osteomyelitis (SBO) is typically bacterial in origin and caused by Pseudomonas, although the fungus Aspergillus has also rarely been implicated . SBO generally arises from ear infections and infrequently complicates sinonasal infection . Rhinocerebral Mucor infection is characteristically an acute, fulminant, and deadly infection also affecting the orbits and deep face and is associated with intracranial complications . Bony involvement is uncommon because of the angioinvasive nature of the fungus . More recently, chronic invasive Mucor sinusitis has been described . We report the unusual clinical and imaging features of a patient with biopsy-proven invasive mucormycosis arising from chronic isolated sphenoid sinus disease, who presented with extensive SBO and a paucity of deep facial, orbital, or intracranial involvement. Biotechnol Appl Biochem, 2000 Jun, 31 ( Pt 3), 179 - 83 Immobilization of Pseudomonas cepacia lipase in a phyllosilicate sol-gel matrix: effectiveness as a biocatalyst; Hsu AF et al.; A novel procedure is described for immobilizing a lipase from Pseudomonas cepacia (PS-30) within a phyllosilicate sol-gel matrix . The method is based on cross-linking a phyllosilicate clay with silicate polymers produced by the controlled hydrolysis of tetramethyl orthosilicate (TMOS) . The activity of the phyllosilicate sol-gel-immobilized lipase was dependent upon the type of alkylammonium salt, inorganic catalyst and volume ratio of phyllosilicate clay to TMOS used . Lipase PS-30 immobilized in this way was more stable and had higher activity compared with the free lipase . Studies on the lipase-catalysed esterification of lauric acid with octan-1-ol in iso-octane showed that under controlled water activity conditions the phyllosilicate sol-gel-immobilized lipase had better activity compared with other supported lipase preparations . In addition, the phyllosilicate sol-gel-immobilized lipase was reusable for at least five esterification cycles without significant loss of activity. EMBO J, 2000 May 15, 19(10), 2257 - 69 Thr38 and Ser198 are Pto autophosphorylation sites required for the AvrPto-Pto-mediated hypersensitive response; Sessa G et al.; The tomato Pto kinase confers resistance to Pseudomonas syringae pv . tomato expressing the AvrPto protein . To elucidate the role of Pto autophosphorylation in disease resistance, eight sites autophosphorylated by Pto in vitro were identified by a combination of HPLC purification of tryptic phosphopeptides, MALDI-TOF/MS analysis and Edman degradation . Mutational analysis of the autophosphorylation sites revealed that Pto residues Thr38 and Ser198 are required for AvrPto-Pto- mediated elicitation of a hypersensitive response in the plant . Thr38, which is the main Pto autophosporylation site and is located outside the kinase catalytic domain, was also required for Pto kinase activity and its physical interaction with AvrPto, the Pti1 kinase and the transcription factor Pti4 . Ser198, located in the Pto activation domain, was dispensable for kinase activity and for interaction with AvrPto . However, a mutation at this site resulted in altered Pto interactions with the Pti1 kinase and the Pto interactors of unknown function Pti3 and Pti10 . These results suggest that autophosphorylation events at Pto Thr38 and Ser198 are required for signal transduction by Pto and participate in distinct molecular mechanisms. Plant Cell, 2000 May, 12(5), 771 - 86 Pti4 is induced by ethylene and salicylic acid, and its product is phosphorylated by the Pto kinase; Gu YQ et al.; The tomato Pti4 gene encodes a transcription factor that was identified on the basis of its specific interaction with the product of the Pto disease resistance gene in a yeast two-hybrid system . We show here that the Pti4 protein specifically binds the GCC-box cis element, which is present in the promoter region of many pathogenesis-related (PR) genes . Expression of the Pti4 gene in tomato leaves was rapidly induced by ethylene and by infection with Pseudomonas syringae pv tomato, and this induction preceded expression of GCC-box-containing PR genes . Although salicylic acid also induced Pti4 gene expression, it did not induce GCC-box PR genes . Rather, salicylic acid antagonized ethylene-mediated expression of GCC-box PR genes . We demonstrate that the Pti4 protein is specifically phosphorylated by the Pto kinase and that this phosphorylation enhances binding of Pti4 to the GCC box . In addition, induced overexpression of Pto and Pti4 in tomato leaves resulted in a concomitant increase in GCC-box PR genes . Our results support a model in which phosphorylation of the Pti4 protein by the Pto kinase enhances the ability of Pti4 to activate expression of GCC-box PR genes in tomato. J Bacteriol, 2000 Jun, 182(11), 3136 - 41 Cloning and expression of ntnD, encoding a novel NAD(P)(+)-independent 4-nitrobenzyl alcohol dehydrogenase from Pseudomonas sp . Strain TW3; James KD et al.; Pseudomonas sp . strain TW3 is able to metabolize 4-nitrotoluene to 4-nitrobenzoate and toluene to benzoate aerobically via a route analogous to the upper pathway of the TOL plasmids . We report the cloning and characterization of a benzyl alcohol dehydrogenase gene (ntnD) which encodes the enzyme for the catabolism of 4-nitrobenzyl alcohol and benzyl alcohol to 4-nitrobenzaldehyde and benzaldehyde, respectively . The gene is located downstream of the previously reported ntn gene cluster . NtnD bears no similarity to the analogous TOL plasmid XylB (benzyl alcohol dehydrogenase) protein either in its biochemistry, being NAD(P)(+) independent and requiring assay via dye-linked electron transfer, or in its deduced amino acid sequence . It does, however, have significant similarity in its amino acid sequence to other NAD(P)(+)-independent alcohol dehydrogenases and contains signature patterns characteristic of type III flavin adenine dinucleotide-dependent alcohol oxidases . Reverse transcription-PCR demonstrated that ntnD is transcribed during growth on 4-nitrotoluene, although apparently not as part of the same transcript as the other ntn genes . The substrate specificity of the enzyme expressed from the cloned and overexpressed gene was similar to the activity expressed from strain TW3 grown on 4-nitrotoluene, providing evidence that ntnD is the previously unidentified gene in the pathway of 4-nitrotoluene catabolism . Examination of the 14.8-kb region around the ntn genes suggests that one or more recombination events have been involved in the formation of their current organization. J Bacteriol, 2000 Jun, 182(11), 3008 - 16 Identification of an effector specificity subregion within the aromatic-responsive regulators DmpR and XylR by DNA shuffling; Skarfstad E et al.; The Pseudomonas derived sigma(54)-dependent regulators DmpR and XylR control the expression of genes involved in catabolism of aromatic compounds . Binding to distinct, nonoverlapping groups of aromatic effectors controls the activities of these transcriptional activators . Previous work has derived a common mechanistic model for these two regulators in which effector binding by the N-terminal 210 residues (the A-domain) of the protein relieves repression of an intrinsic ATPase activity essential for its transcription-promoting property and allows productive interaction with the transcriptional apparatus . Here we dissect the A-domains of DmpR and XylR by DNA shuffling to identify the region(s) that mediates the differences in the effector specificity profiles . Analysis of in vivo transcription in response to multiple aromatic effectors and the in vitro phenol-binding abilities of regulator derivatives with hybrid DmpR/XylR A-domains reveals that residues 110 to 186 are key determinants that distinguish the effector profiles of DmpR and XylR . Moreover, the properties of some mosaic DmpR/XylR derivatives reveal that high-affinity aromatic effector binding can be completely uncoupled from the ability to promote transcription . Hence, novel aromatic binding properties will only be translated into functional transcriptional activation if effector binding also triggers release of interdomain repression. Plant Cell Physiol, 2000 Mar, 41(3), 327 - 34 Ethylene formation and phenotypic analysis of transgenic tobacco plants expressing a bacterial ethylene-forming enzyme; Araki S et al.; A bacterial ethylene-forming enzyme (EFE) catalyzes oxygenation of 2-oxoglutarate to produce ethylene and carbon dioxide in contrast to a plant enzyme which uses 1-aminocyclopropane-1-carboxylic acid as a substrate . We constructed several lines of transgenic tobacco plants which expressed an EFE from Pseudomonas syringae pv . phaseolicola PK2 . The gene encoding a chimeric protein consisting of EFE and beta-glucuronidase (GUS) was introduced into the tobacco genome using a binary vector which directs expression of the EFE-GUS fusion protein under the control of constitutive promoter of cauliflower mosaic virus 35S RNA . Two lines of transgenic plants produced ethylene at consistently higher rates than the untransformed plant, and their GUS activities were expressed in different tissues . A significant dwarf morphology observed in the transgenic tobacco displaying the highest ethylene production resembled the phenotype of a wild-type plant exposed to excess ethylene . These results demonstrate a potential use of bacterial EFE to supply ethylene as a hormonal signal via an alternative route using an ubiquitous substrate 2-oxoglutarate in plant tissues. Org Lett, 2000 Apr 20, 2(8), 1089 - 92 Synthesis of unnatural amino acids via Suzuki cross-coupling of enantiopure vinyloxazolidine derivatives; Sabat M et al.; {formula: see text} (R and S)-alpha-Amino alcohols and alpha-amino acids, including 4-methoxyhomophenylalanine, with a variety of unnatural side chains have been synthesized via palladium-catalyzed cross-coupling Suzuki reactions . The key building blocks 1 and 2, synthesized from the common achiral precursor 2-butene-1,4-diol, were made enantiopure utilizing a Pseudomonas cepacia lipase-catalyzed kinetic resolution . The optimal conditions for the Suzuki cross-coupling and the subsequent oxidations of the resultant alpha-amino alcohols are described. Org Lett, 2000 Apr 20, 2(8), 1037 - 40 Dynamic kinetic resolution of alpha-hydroxy acid esters Huerta FF, Laxmi YR, Backvall JE. {formula: see text} Enzymatic resolution in combination with ruthenium-catalyzed racemization of the substrate led to dynamic kinetic resolution of alpha-hydroxy esters in good yields and excellent ee's . Studies of different parameters showed that the best results were obtained using Pseudomonas cepacia lipase, ruthenium catalyst 3, and 4-chlorophenyl acetate as acyl donor in cyclohexane. Appl Microbiol Biotechnol, 2000 Apr, 53(4), 401 - 9 Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant bacteria expressing the PHA synthase gene phaC1 from Pseudomonas sp . 61-3; Matsusaki H et al.; Pseudomonas sp . 61-3 accumulated a blend of poly(3-hydroxybutyrate) {P(3HB)} homopolymer and a random copolymer consisting of 3-hydroxyalkanoate (3HA) units of 4-12 carbon atoms . The genes encoding beta-ketothiolase (PhbA(Re)) and NADPH-dependent acetoacetyl-CoA reductase (PhbB(Re)) from Ralstonia eutropha were expressed under the control of promoters for Pseudomonas sp . 61-3 pha locus or R . eutropha phb operon together with phaC1(Ps) gene (PHA synthase 1 gene) from Pseudomonas sp . 61-3 in PHA-negative mutants P . putida GPp104 and R . eutropha PHB(-4) to produce copolyesters {P(3HB-co-3HA)} consisting of 3HB and medium-chain-length 3HA units of 6-12 carbon atoms . The introduction of the three genes into GPp104 strain conferred the ability to synthesize P(3HB-co-3HA) with relatively high 3HB compositions (up to 49 mol%) from gluconate and alkanoates, although 3HB units were not incorporated at all or at a very low fraction (3 mol%) into copolyesters by the strain carrying phaC1Ps gene only . In addition, recombinant strains of R . eutropha PHB(-4) produced P(3HB-co-3HA) with higher 3HB fractions from alkanoates and plant oils than those from recombinant GPp104 strains . One of the recombinant strains, R . eutropha PHB(-4)/ pJKSc46-pha, in which all the genes introduced were expressed under the control of the native promoter for Pseudomonas sp . 61-3 pha locus, accumulated P(3HB-co-3HA) copolyester with a very high 3HB fraction (85 mol%) from palm oil . The nuclear magnetic resonance analyses showed that the copolyesters obtained here were random copolymers of 3HB and 3HA units. J Biol Chem, 2000 May 12, 275(19), 14375 - 80 Conversion of interleukin-13 into a high affinity agonist by a single amino acid substitution; Oshima Y et al.; We created a novel mutated form of human interleukin-13 (IL-13) in which a positively charged arginine (R) at position 112 was substituted to a negatively charged aspartic acid (D) . This mutant, termed IL-13R112D, was expressed in Escherichia coli and purified to near homogeneity . IL-13R112D was found to be a potent IL-13 agonist with 5-10-fold improved binding affinity to IL-13 receptors compared with wild-type IL-13 (wtIL-13) . The conclusion of IL-13 agonist activity was drawn on the basis of approximately 10-fold improved activity over wtIL-13 in several assays: (a) inhibition of CD14 expression in primary monocytes; (b) proliferation of TF-1 and B9 cell lines; and (c) activation of STAT6 in Epstein-Barr virus-immortalized B cells, primary monocytes, and THP-1 monocytic cell line . Furthermore, mutant IL-13R112D neutralized the cytotoxic activity of a chimeric fusion protein composed of wtIL-13 and a Pseudomonas exotoxin A (IL-13-PE38) approximately 10 times better than wtIL-13 . Based on these results, it was concluded that IL-13R112D interacts with much stronger affinity than wtIL-13 on all cell types tested and that Arg-112 plays an important role in the interaction with its receptors (IL-13R) . Thus, these results suggest that IL-13R112D may be a useful ligand for the study of IL-13 interaction with its receptors or, alternatively, in designing specific targeted agents for IL-13R-positive malignancies. Int J Cancer, 2000 Jun 1, 86(5), 718 - 24 Recombinant anti-CD25 immunotoxin RFT5(SCFV)-ETA' demonstrates successful elimination of disseminated human Hodgkin lymphoma in SCID mice; Barth S et al.; Since clinical phase-I/II trials in patients with resistant Hodgkin's lymphoma treated with the chemically linked anti-CD25 ricin-A-chain immunotoxin RFT5-SMPT-dgA indicate promising results for patients with minimal residual disease, we constructed a new immunotoxin by fusing the RFT5 single-chain variable fragment to a deletion mutant of Pseudomonas exotoxin A (ETA') . The recombinant protein was directed into the periplasmic space of E . coli by means of the pET-derived expression vector pBM1.1 and our newly developed expression/purification method . Biologically active RFT5(scFv)-ETA' was isolated by freezing/thawing and purified by immobilized metal-ion affinity and molecular-size-chromatography . RFT5(scFv)-ETA' was subsequently used for the treatment of disseminated human Hodgkin's lymphoma in a SCID-mouse model . The mean survival time (MST) of L540rec-challenged SCID mice was 38.1 days . A single i.v . injection of 40 microg recombinant immunotoxin (rIT) 1 day after tumor inoculation resulted in 100% tumor-free mice, extending the MST to more than 220 days (p < 0.0001) . The blood-distribution time T(1/2)alpha was 39.65 min, the serum elimination time T(1/2)alpha, 756.6 min . All animals were assessed for soluble interleukin-2 receptor alpha, which is directly correlated to tumor burden . Soluble CD25 was not detectable in mice treated with the rIT . Our findings, concerning potent anti-tumor effects of a recombinant anti-CD25 immunotoxin against disseminated Hodgkin's lymphoma in SCID mice reported here demonstrate that RFT5(scFv)-ETA' might be suitable for further evaluation against Hodgkin's lymphoma in humans . Mol Plant Microbe Interact, 2000 May, 13(5), 568 - 71 avrPto enhances growth and necrosis caused by Pseudomonas syringae pv.tomato in tomato lines lacking either Pto or Prf; Chang JH et al.; AvrPto was introduced into three tomato genotypes with two biotic agents to study its role in compatible interactions . avrPto enhanced the capacity of the Pseudomonas syringae pv . tomato strain T1 to induce necrotic symptoms on tomato plants that lacked either Pto or Prf genes . The enhanced necrosis correlated with a small increase in bacterial growth . In planta expression of avrPto in isolation did not elicit necrosis in the absence of a functional Prf gene. Mol Plant Microbe Interact, 2000 May, 13(5), 563 - 7 Decreased inositol 1,4,5-trisphosphate content in pathogen-challenged soybean cells; Shigaki T et al.; Phosphoinositide-specific phospholipase C (PI-PLC) has been shown to be transiently activated when plant cells were treated with elicitors . We thus investigated the activity of PI-PLC when soybean cells were infected with the bacterial pathogen Pseudomonas syringae pv . glycinea, by measuring cellular cytosolic inositol 1,4,5-trisphosphate (IP3) levels . We observed that IP3 content decreased in both compatible and incompatible interactions . In vitro phosphatase activities were similar in both water control and infected cells with slightly lower IP3 degradation observed for infected cells, indicating that the reduced IP3 content in infected cells most likely results from reduced PI-PLC activity . We hypothesize that reduced IP3 content following infection may lead to suppression of various housekeeping activities of the cells, thus diverting the cellular resources either to the synthesis of defense-related compounds against pathogens, and/or to the growth of pathogens. Mol Plant Microbe Interact, 2000 May, 13(5), 503 - 11 Arabidopsis thaliana EDS4 contributes to salicylic acid (SA)-dependent expression of defense responses: evidence for inhibition of jasmonic acid signaling by SA; Gupta V et al.; The Arabidopsis enhanced disease susceptibility 4 (eds4) mutation causes enhanced susceptibility to infection by the bacterial pathogen Pseudomonas syringae pv . maculicola ES4326 (Psm ES4326) . Gene-for-gene resistance to bacteria carrying the avirulence gene avrRpt2 is not significantly affected by eds4 . Plants homozygous for eds4 exhibit reduced expression of the pathogenesis-related gene PR-1 after infection by Psm ES4326, weakened responses to treatment with the signal molecule salicylic acid (SA), impairment of the systemic acquired resistance response, and reduced accumulation of SA after infection with Psm ES4326 . These phenotypes indicate that EDS4 plays a role in SA-dependent signaling . SA has been shown to have a negative effect on activation of gene expression by the signal molecule jasmonic acid (JA) . Two mutations that cause reduced SA levels, eds4 and pad4, cause heightened responses to inducers of JA-dependent gene expression, providing genetic evidence to support the idea that SA interferes with JA-dependent signaling . Two possible working models of the role of EDS4 in governing activation of defense responses are presented. Appl Environ Microbiol, 2000 May, 66(5), 2085 - 95 Molecular and phenotypic characterization of Pseudomonas spp . isolated from milk; Wiedmann M et al.; Putative Pseudomonas spp . isolated predominantly from raw and processed milk were characterized by automated ribotyping and by biochemical reactions . Isolates were biochemically profiled using the Biolog system and API 20 NE and by determining the production of proteases, lipases, and lecithinases for each isolate . Isolates grouped into five coherent clusters, predominated by the species P . putida (cluster A), P . fluorescens (cluster B), P . fragi (as identified by Biolog) or P . fluorescens (as identified by API 20 NE) (cluster C), P . fragi (as identified by Biolog) or P . putida (as identified by API 20 NE) (cluster D), and P . fluorescens (cluster E) . Isolates within each cluster also displayed similar enzyme activities . Isolates in clusters A, C, and D were generally negative for all three enzyme activities; isolates in cluster B were predominantly positive for all three enzyme activities; and isolates in cluster E were negative for lecithinase but predominantly positive for protease and lipase activities . Thus, only isolates from clusters B and E produced enzyme activities associated with dairy product flavor defects . Thirty-eight ribogroups were differentiated among the 70 isolates . Ribotyping was highly discriminatory for dairy Pseudomonas isolates, with a Simpson's index of discrimination of 0.955 . Isolates of the same ribotype were never classified into different clusters, and ribotypes within a given cluster generally showed similar ribotype patterns; thus, specific ribotype fragments may be useful markers for tracking the sources of pseudomonads in dairy production systems . Our results suggest that ribogroups are generally homogeneous with respect to nomenspecies and biovars, confirming the identification potential of ribotyping for Pseudomonas spp. Appl Environ Microbiol, 2000 May, 66(5), 1939 - 46 Genotypic and phenotypic diversity of phlD-containing Pseudomonas strains isolated from the rhizosphere of wheat; McSpadden Gardener BB et al.; Production of 2,4-diacetylphloroglucinol (2,4-DAPG) in the rhizosphere by strains of fluorescent Pseudomonas spp . results in the suppression of root diseases caused by certain fungal plant pathogens . In this study, fluorescent Pseudomonas strains containing phlD, which is directly involved in the biosynthesis of 2,4-DAPG, were isolated from the rhizosphere of wheat grown in soils from wheat-growing regions of the United States and The Netherlands . To assess the genotypic and phenotypic diversity present in this collection, 138 isolates were compared to 4 previously described 2, 4-DAPG producers . Thirteen distinct genotypes, one of which represented over 30% of the isolates, were differentiated by whole-cell BOX-PCR . Representatives of this group were isolated from eight different soils taken from four different geographic locations . ERIC-PCR gave similar results overall, differentiating 15 distinct genotypes among all of the isolates . In most cases, a single genotype predominated among isolates obtained from each soil . Thirty isolates, representing all of the distinct genotypes and geographic locations, were further characterized . Restriction analysis of amplified 16S rRNA gene sequences revealed only three distinct phylogenetic groups, one of which accounted for 87% of the isolates . Phenotypic analyses based on carbon source utilization profiles revealed that all of the strains utilized 49 substrates and were unable to grow on 12 others . Individually, strains could utilize about two-thirds of the 95 substrates present in Biolog SF-N plates . Multivariate analyses of utilization profiles revealed phenotypic groupings consistent with those defined by the genotypic analyses. Appl Environ Microbiol, 2000 May, 66(5), 1877 - 82 Toluene monooxygenase-catalyzed epoxidation of alkenes; McClay K et al.; Several toluene monooxygenase-producing organisms were tested for their ability to oxidize linear alkenes and chloroalkenes three to eight carbons long . Each of the wild-type organisms degraded all of the alkenes that were tested . Epoxides were produced during the oxidation of butene, butadiene, and pentene but not hexene or octadiene . A strain of Escherichia coli expressing the cloned toluene-4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 was able to oxidize butene, butadiene, pentene, and hexene but not octadiene, producing epoxides from all of the substrates that were oxidized . A T4MO-deficient variant of P . mendocina KR1 oxidized alkenes that were five to eight carbons long, but no epoxides were detected, suggesting the presence of multiple alkene-degrading enzymes in this organism . The alkene oxidation rates varied widely (ranging from 0 . 01 to 0.33 micromol of substrate/min/mg of cell protein) and were specific for each organism-substrate pair . The enantiomeric purity of the epoxide products also varied widely, ranging from 54 to >90% of a single epoxide enantiomer . In the absence of more preferred substrates, such as toluene or alkenes, the epoxides underwent further toluene monooxygenase-catalyzed transformations, forming products that were not identified. Planta, 2000 Mar, 210(4), 563 - 73 Leucine aminopeptidases: the ubiquity of LAP-N and the specificity of LAP-A; Chao WS et al.; The wound-induced leucine aminopeptidase (EC 3.4.11.1) genes, LapA1 and LapA2, from tomato (Lycopersicon esculentum Mill.) were isolated and characterized . The genes were organized in a tandem array with approximately 6 kb separating their coding regions . Quantitation of LapA mRNA levels in conjunction with nuclear run-on experiments indicated that LapA genes were primarily under transcriptional control after wounding and infection with Pseudomonas syringae pv . tomato . In contrast, actin genes were down-regulated after pathogen infection . The sequences of the LapA1 and LapA2 5'-flanking regions were determined and several potential regulatory motifs were identified . Ribonuclease protection studies revealed that LapA1 and LapA2 had short 18-bp 5'-untranslated regions (UTR), both genes were expressed after wounding, and LapA1 mRNAs were 3.3-fold more abundant than LapA2 transcripts . While the region surrounding LapA1 was conserved, the 3'-UTRs and 3'-flanking regions of LapA2 had diverged in two inbred tomato lines . The accumulation of LapA mRNAs and of LAP-A (acidic pI), LAP-N (neutral pI) and LAP-related proteins were examined in two monocot and five dicot species . The LAP-N and 66- and 77-kDa LAP-related proteins were detected in healthy and wounded leaves of all plants examined . The LAP-A proteins were only detected in nightshade and their accumulation was distinct from that observed in tomato. Mol Med, 1998 Jun, 4(6), 384 - 91 Specific killing of HIV-infected lymphocytes by a recombinant immunotoxin directed against the HIV-1 envelope glycoprotein; Bera TK et al.; BACKGROUND: 3B3 is a high-affinity anti-gp120 antibody that neutralizes a wide range of primary and laboratory isolates of HIV-1 . The parental antibody was isolated from a combinatorial phage display library constructed from bone marrow RNA of an HIV-infected individual . We have generated a highly active immunotoxin using the 3B3 single-chain Fv (scFv) which can specifically kill lymphocytes infected by HIV-1 . MATERIALS AND METHODS: We used recombinant DNA technology to clone the Fv fragment of 3B3 and produce a single-chain Fv (scFv) . 3B3 scFv was then fused to a truncated version of Pseudomonas exotoxin A (PE38), giving rise to a recombinant immunotoxin 3B3(Fv)-PE38 that was expressed in E . coli and purified to near homogeneity . RESULTS: 3B3(Fv)-PE38 binds with the same affinity as the parental Fab antibody to the MN strain of gp120 . The immunotoxin specifically kills a gp120-expressing transfected cell line and a chronically HIV-infected lymphocytic cell line . The immunotoxin is very stable at 37 degrees C, retaining 80% of its original activity after 24 hr . CONCLUSIONS: Potent immunotoxins such as 3B3(Fv)-PE38 could be utilized in combination with multidrug cocktails that limit viral replication to help reduce viral reservoirs in patients with AIDS. Clin Cancer Res, 2000 Apr, 6(4), 1476 - 87 Cytotoxic activity of disulfide-stabilized recombinant immunotoxin RFB4(dsFv)-PE38 (BL22) toward fresh malignant cells from patients with B-cell leukemias; Kreitman RJ et al.; Chemical conjugates of anti-CD22 monoclonal antibodies and toxins have been used to treat CD22+ hematological malignancies . A new anti-CD22 recombinant immunotoxin RFB4(dsFv)-PE38, composed of the Fv portion of the monoclonal antibody RFB4 fused to a truncated form of Pseudomonas exotoxin A, is being developed to target CD22+ tumor cells . To explore the potential clinical utility of this recombinant toxin in treating patients with B-cell malignancies, the fresh cells of patients were incubated ex vivo with RFB4(dsFv)-PE38 . Specific cytotoxicity was demonstrated in the malignant cells of 25 of 28 patients with a variety of B-cell malignancies, including acute and chronic lymphocytic leukemias and large cell, mantle cell, and follicular lymphomas . The IC50S, the concentrations necessary for 50% inhibition of protein synthesis, were 3-10 ng/ml in five patients and 10-50 ng/ml in seven patients . Cytotoxicity correlated with cell death upon direct examination of the malignant cells . Significant cytotoxicity was observed with cells containing as few as 350 CD22 sites/cell . A more active derivative of RFB4(dsFv)-PE38, RFB4(dsFv)-PE38KDEL, was produced and was slightly to more than 10-fold more cytotoxic toward patient cells and about twice as toxic to mice . Thus, RFB4(dsFv)-PE38 was specifically cytotoxic toward malignant cells from patients with B-cell leukemias . These data support the testing of RFB4(dsFv)-PE38 in patients with CD22+ leukemias and lymphomas, which is presently under way. J Pediatr Gastroenterol Nutr, 2000 Apr, 30(4), 426 - 31 Seroprevalence of Helicobacter pylori infection in cystic fibrosis and its cross-reactivity with anti-pseudomonas antibodies; Israel NR et al.; BACKGROUND: The prevalence of Helicobacter pylori infection and its role in gastroduodenal disease in cystic fibrosis (CF) are controversial . Additionally, serologic determination of infection in this population may be inaccurate because of cross-reactivity with other bacterial species . The seroprevalence of H . pylori in a cohort of patients with CF and its cross-reactivity with Pseudomonas antibodies were investigated . METHODS: A research enzyme-linked immunosorbent assay (ELISA), and three commercial serologic assays (PyloriStat; BioWhittaker, Walkersville, MD, U.S.A.; Flexsure; SmithKline Diagnostics, Inc., San Jose, CA, U.S.A.; and HM-CAP; EPI, Stony Brook, NY, U.S.A.) at three independent laboratories determined the seroprevalence of anti-H . pylori IgG antibodies in 70 patients with CF . Cross-reactivity between solid-phase H . pylori antigens and Pseudomonas antibodies was ascertained by a competitive inhibition assay, preadsorbing sera of patients with CF with whole cell proteins from different Pseudomonas species, and serum reanalysis by each assay . Western blot analysis before and after adsorption was performed to identify potential cross-reactive antigens . RESULTS: The research ELISA, Flexsure, Pyloristat, and HM-CAP initially showed H . pylori seropositivity of 47%, 28%, 24%, and 37%, respectively . Postadsorption seropositivity declined to 8%, 0%, 0%, and 15%, respectively . All patients with research ELISA true-positive results were confirmed endoscopically to have H . pylori infection . Western blot analysis showed a 31-kDa H . pylori protein with antigenic epitopes common to both bacterial species . CONCLUSIONS: Cross-reactivity between solid-phase H . pylori antigens and anti-Pseudomonas antibodies occurs in patients with CF . A high index of suspicion should be assumed in evaluating results of serologic H . pylori tests in this population . Preadsorption of CF sera with Pseudomonas proteins should be used in serologic testing. IUBMB Life, 2000 Feb, 49(2), 137 - 41 Vanadium-binding protein excreted by vanadate-reducing bacteria; Antipov AN et al.; A vanadium-binding protein was isolated from the culture medium of the vanadium-reducing bacterium Pseudomonas isachenkovii by utilizing vanadate as the terminal electron acceptor upon anaerobic respiration . The protein was associated with vanadium at a molar ratio of approximately 1:20 . It was purified to homogeneity and separated into three components by treatment with 1 M HCl followed by gel filtration: a protein, a vanadium-binding ligand, and inorganic vanadium . Electron paramagnetic resonance analysis showed that vanadium was associated with the protein in the 4+ oxidation state . The distribution of vanadium within the cell was studied by electron microscopy and x-ray microanalysis of P . isachenkovii cells . The results suggest that vanadium, accumulated in special swells on the surface of the cell membranes, is reduced and excreted to the medium. Thorax, 2000 May, 55(5), 355 - 8 Elective versus symptomatic antibiotic treatment in cystic fibrosis patients with chronic Pseudomonas infection of the lungs; Elborn JS et al.; BACKGROUND: A previous retrospective study suggested that a policy of regular anti-pseudomonal antibiotic treatment improved pulmonary function and increased survival in patients with cystic fibrosis chronically infected with Pseudomonas species . The results of a prospective multicentre study to compare the effects on pulmonary function and mortality of three monthly elective anti-pseudomonal antibiotic treatment with conventional symptomatic treatment are reported . METHODS: Sixty patients with cystic fibrosis, chronically infected with P aeruginosa, were randomised to the two treatment arms (elective or symptomatic) and followed clinically at yearly reviews . The major end points were changes in forced expiratory volume in one second (FEV(1)) and forced vital capacity (FVC) . Survival was a secondary end point . RESULTS: Patients in the symptomatic group received a mean of three antibiotic treatments each year and those in the elective group received four antibiotic treatments during each year of the study . No significant differences in FEV(1) and FVC were found between the two groups after three years . There was a statistically non-significant higher rate of deaths in the elective group (n = 4), three of which were associated with B cepacia infection, compared with the symptomatic group (n = 0) . CONCLUSIONS: This study did not demonstrate an advantage of a policy of elective antibiotic treatment over symptomatic treatment in patients with cystic fibrosis chronically infected with Pseudomonas species. J Clin Oncol, 2000 Apr, 18(8), 1622 - 36 Phase I trial of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) in patients with hematologic malignancies; Kreitman RJ et al.; PURPOSE: To evaluate the toxicity, pharmacokinetics, immunogenicity, and antitumor activity of anti-Tac(Fv)-PE38 (LMB-2), an anti-CD25 recombinant immunotoxin that contains an antibody Fv fragment fused to truncated Pseudomonas exotoxin . PATIENTS AND METHODS: Patients with CD25(+) hematologic malignancies for whom standard and salvage therapies failed were treated with LMB-2 at dose levels that ranged from 2 to 63 microg/kg administered intravenously over 30 minutes on alternate days for three doses (QOD x 3) . RESULTS: LMB-2 was administered to 35 patients for a total of 59 cycles . Dose-limiting toxicity at the 63 microg/kg level was reversible and included transaminase elevations in one patient and diarrhea and cardiomyopathy in another . LMB-2 was well tolerated in nine patients at the maximum-tolerated dose (40 microg/kg QOD x 3); toxicity was transient and most commonly included transaminase elevations (eight patients) and fever (seven patients) . Only six of 35 patients developed significant neutralizing antibodies after the first cycle . The median half-life was 4 hours . One hairy cell leukemia (HCL) patient achieved a complete remission, which is ongoing at 20 months . Seven partial responses were observed in cutaneous T-cell lymphoma (one patient), HCL (three patients), chronic lymphocytic leukemia (one patient), Hodgkin's disease (one patient), and adult T-cell leukemia (one patient) . Responding patients had 2 to 5 log reductions of circulating malignant cells, improvement in skin lesions, and regression of lymphomatous masses and splenomegaly . All four patients with HCL responded to treatment . CONCLUSION: LMB-2 has clinical activity in CD25(+) hematologic malignancies and is relatively nonimmunogenic . It is the first recombinant immunotoxin to induce major responses in cancer . LMB-2 and similar agents that target other cancer antigens merit further clinical development. J Bacteriol, 2000 May, 182(9), 2624 - 8 The GacS sensor kinase regulates alginate and poly-beta-hydroxybutyrate production in Azotobacter vinelandii; Castaneda M et al.; Azotobacter vinelandii produces two polymers: the extracellular polysaccharide alginate and the intracellular polyester poly-beta-hydroxybutyrate (PHB) . A cosmid clone (pSMU588) from an A . vinelandii gene library diminished alginate production by A . vinelandii mucoid strain ATCC 9046 . The nucleotide sequence and predicted amino acid sequence of the locus responsible for the mucoidy suppression revealed 65% identity to Pseudomonas GacS, a transmembrane sensor kinase of the two-component regulators, whose cognate response regulator, GacA, is a global activator regulating several products and virulence factors . Plasmid pMC15, harboring gacS, and a strain carrying a gacS nonpolar mutation were constructed . Either pMC15 or the gacS mutation significantly reduced alginate production and transcription of algD, the gene coding for the key enzyme GDP-mannose dehydrogenase of the alginate biosynthetic pathway . We found that the gacS mutation also reduced PHB accumulation and impaired encystment . Taken together, these data indicate that in A . vinelandii the gacSA global system regulates polymer synthesis. Eur J Biochem, 2000 Apr, 267(8), 2372 - 9 Structure of the O polysaccharide and serological classification of Pseudomonas syringae pv . ribicola NCPPB 1010; Ovod VV et al.; The O polysaccharide (OPS) moiety of the lipopolysaccharide (LPS) of a phytopathogenic bacterium Pseudomonas syringae pv . ribicola NCPPB 1010 was studied by sugar and methylation analyses, Smith degradation, and 1H- and 13C-NMR spectroscopy, including 2D COSY, TOCSY, NOESY and H-detected 1H,13C HMQC experiments . The OPS structure was elucidated, and shown to be composed of branched pentasaccharide repeating units (O repeats) of two types, major (1) and minor (2), differing in the position of substitution of one of the rhamnose residues . Both O repeats form structurally homogeneous blocks within the same polysaccharide molecule . Although P . syringae pv . ribicola NCPPB 1010 demonstrates genetic relatedness and similarity in the OPS chemical structure to some other P . syringae pathovars, it did not cross-react with any OPS-specific mAbs produced against heterologous P . syringae strains . Therefore, we propose to classify P . syringae pv . ribicola NCPPB 1010 in a new serogroup, O8. Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 711 - 3 Taxonomic implications of synthesis of poly-beta-hydroxybutyrate and other poly-beta-hydroxyalkanoates by aerobic pseudomonads; Kessler B et al.; Whereas poly-beta-hydroxybutyrate (PHB) production by Pseudomonas species is rare, synthesis of medium-chain-length poly-beta-hydroxyalkanoates (mcl-PHAs) other than PHB, has been observed in fluorescent and non-fluorescent species . Contrary to original reports, Pseudomonas corrugata and Pseudomonas ficuserectae accumulate mcl-PHAs and not PHB . The taxonomic implications of these characteristics are discussed. Plant J, 2000 Feb, 21(4), 393 - 9 AtBI-1, a plant homologue of Bax inhibitor-1, suppresses Bax-induced cell death in yeast and is rapidly upregulated during wounding and pathogen challenge; Sanchez P et al.; Extensive searches have so far failed to identify functional plant homologues of the mammalian apoptotic machinery . Here we report the isolation and characterisation of an Arabidopsis thaliana homologue of human Bax Inhibitor-1, AtBI-1, isolated during a differential screen of plants challenged with the phytopathogen Pseudomonas syringae . AtBI is a member of a small gene family in Arabidopsis, members of which display extensive amino acid identity to human BI-1 . AtBI-1 is also functionally similar to BI-1 in its ability to suppress the lethal phenotype in yeast conferred by expression of the mammalian proapoptotic protein, Bax . Expression of AtBI-1 is rapidly upregulated in plants during wounding or pathogen challenge, suggesting a role in responses to biotic and abiotic stress . AtBI-1 upregulation appears R gene independent and is not markedly affected by mutations required for specific classes of R genes . However, the accumulation of AtBI-1 message is significantly reduced in coi1, in which defence responses to insects, pathogens and wounding are compromised. In Vivo, 2000 Jan-Feb, 14(1), 21 - 7 Recombinant antibody fragments and immunotoxin fusions for cancer therapy; Brinkmann U; Recombinant immunotoxins consist of Fv regions of cancer specific antibodies fused to truncated bacterial toxins . Many recombinant immunotoxins contain a truncated version of Pseudomonas Exotoxin as a toxic moiety . This toxin is modified in such a manner that by itself it does not bind to normal human cells, but it retains all other functions of cytotoxicity . The recombinant antibody fragments target the modified toxin to cancer cells which are killed, either by direct inhibition of protein synthesis, or by concomitant induction of apoptosis . Cells that are not recognized by the antibody fragment, because they do not carry the cancer antigen, are spared . Many factors influence the in vivo anti-tumor activity of recombinant immunotoxins . Among them are considerations of which types of cancer and at what stages may be the best targets for immunotoxin therapy and tumor specificity of the antigen that is targeted by the recombinant antibody . Also the affinity of immunotoxins and their ability to enter and penetrate into tissues and tumors, which in turn is dependent on the size of the protein . And, because one very important factor is the stability of immunotoxins, a great deal of protein-engineering is required to stabilize the recombinant antibody moiety of immunotoxins . Excellent activity and specificity can be observed for many recombinant immunotoxins in in vitro assays using cultured cancer cells as well as in animal tumor models . Ongoing clinical trials provide examples where the promising preclinical data correlate with successful results in experimental cancer therapy. FEMS Microbiol Lett, 2000 Apr 15, 185(2), 163 - 8 The polyester polyurethanase gene (pueA) from Pseudomonas chlororaphis encodes a lipase; Stern RV et al.; A gene (pueA, polyurethane esterase A) encoding an extracellular polyurethanase (PueA) was cloned from Pseudomonas chlororaphis into Escherichia coli . The enzyme secreted from E . coli showed esterase activity when assayed with p-nitrophenyl acetate . Subcloning of a 3 . 2-kb SalI-EcoRI fragment into a T7 RNA polymerase expression vector (pT7-6) produced a (35)S-labeled protein of 65 kDa . Nucleotide sequencing of pueA showed an open reading frame encoding a 65-kDa protein of 617 amino acid residues, with the serine hydrolase consensus sequence GXSXG . PueA was over-expressed using the pT7-6 vector transformed into E . coli BL21(DE3) and was purified in one step using Sephadex G-75. Biotechnol Prog, 2000 Mar-Apr, 16(2), 287 - 91 Covalent and metal-chelate immobilization of a modified 2-haloacid dehalogenase for the enzymatic resolution of optically active chloropropionic acid; Ordaz E et al.; The stereospecific L-2-haloacid dehalogenase DehCI from Pseudomonas CBS3 was tagged with a peptide tail containing six histidines and overexpressed in Escherichia coli . The His-tagged protein was purified after a single-step affinity chromatography on Zn(2+)-chelating sepharose . The activity of the modified protein was tested after immobilization on Zn(2+)-chelating sepharose and on covalently bound acrylic polymer . Both immobilization systems were used for the transformation of racemic 2-chloropropionic acid into D-lactate and D-chloropropionic acid . Although immobilization on chelating sepharose produced a limited increase in stability, covalent immobilization on acrylic polymer significantly extended the operational temperature and pH range of the enzyme: up to 60% of activity was recovered at either 80 degrees C or pH 11, whereas no activity could be detected under these conditions in the soluble or chelate-immobilized enzyme . Both forms of immobilization extended the enzyme effective storage periods, and after 10 cycles of reutilization, 70% and 20% of the initial activity was recovered in the covalent- and chelate-immobilized enzyme, respectively. Prikl Biokhim Mikrobiol, 2000 Jan-Feb, 36(1), 55 - 8 {Isolation of highly active strain producing the antistaphylococcal antibiotic batumin}; Smirnov VV et al.; The use of chemical and UV-induced mutageneses allowed us to increase the biosynthetic activity of the strain capable of producing new antistaphylococcal antibiotic, batumin . The strain of Pseudomonas batumici N17 producing 87-100 mg batumin per liter culture liquid was selected . Its activity was 3.5-5 times higher than the activity of the most potent natural strain . P . batumici N17 was shown to be stable in relation to the synthesis of batumin. Genetika, 2000 Feb, 36(2), 159 - 64 {Mutants of the nitrogen-fixing rhizospheric bacteria Pseudomonas sp . 418 with loss of the ability to colonize roots}; Bazhanov DP et al.; Mutants of nitrogen-fixing rhizospheric bacterium Pseudomonas sp . 418, which lacked the competitive ability to colonize roots, were induced by random Tn5 mutagenesis . By means of Southern blot analysis, it was shown that single transposon insertions occurred in eight mutants, whereas in two mutants, the Tn5 insertion occurred twice in different DNA regions . Analysis of these mutants revealed the following disturbances of characters having adaptive significance: weakened attachment to the root surface; a defect in chemotaxis; impaired motility as a result of the loss of flagella or nondisjunction of cells after division; and alterations in the synthesis of exopolysaccharides . The effect of Tn5 insertions was, as a rule, pleiotropic, which suggests the coordinated expression of traits essential for the survival of bacterial cells in the root region. Can J Microbiol, 2000 Mar, 46(3), 259 - 68 Adhesion to hyphal matrix and antifungal activity of Pseudomonas strains isolated from Tuber borchii ascocarps; Sbrana C et al.; Pseudomonas spp . isolates from Tuber borchii ascocarps, known to be able to produce phytoregulatory and biocontrol substances in pure culture, were used to perform studies on their possible physiological role in nature . Antimycotic activity was confirmed against fungal contaminants isolated from the ascocarps, suggesting that populations associated with Tuber borchii fruit bodies may play a role in the maintenance of ascocarp health . Fifty-five percent of strains tested were also able to release metabolites which affected T . borchii mycelial growth and morphogenesis in culture . On the contrary, growth of the arbuscular mycorrhizal fungus Glomus mosseae and the ectomycorrhizal fungus Laccaria bicolor, putative competitors of Tuber for mycorrhizal infection sites on roots, was not influenced by the presence of any bacterial strain . The possibility that these bacteria, which show antifungal activity and fungal growth modulation activities, might be incorporated in the developing ascocarp by means of their preferential adhesion to Tuber mycelium is discussed. Can J Microbiol, 2000 Mar, 46(3), 246 - 58 Aspects of the pathology and etiology of 'drippy gill' disease of the cultivated mushroom Agaricus bisporus; Gill W et al.; Agaricus bisporus sporocarps exhibiting characteristic 'drippy gill' symptoms from a natural outbreak were examined . Discrete bacterial droplets on the hymenial lamellae often coalesced to form ribbons of bacterial ooze . Longitudinal splits on the stipe were lined with a similar bacterial ooze . Bacteria isolated from both the hymenium and stipe were identified as Pseudomonas agarici, and were confirmed to be the causal organism by satisfying Koch's postulates . By light and transmission electron microscopy, the causal bacteria were found to colonize the extrahyphal spaces and degrade the extracellular matrix within affected sporocarps . Degradation of the extracellular matrix was shown to reduce the integrity of the sporocarp, and result in stipe splitting and hymenium disruption . In artificial inoculations of the pileus, bacteria were shown to exist predominantly in sporocarp tissue below the point of inoculation and above affected areas of the hymenium, indicating an approximately vertical passage through the sporocarp via the extracellular spaces . The dissolution of the extracellular matrix, and the observed failure of the bacterium to produce a toxin active against A . bisporus, allow the bacteria to pass through protective membranes unnoticed, and infect the stipe and hymenium prior to veil break . These observations dispel previous assumptions of intrahyphal existence and transmission . In the few instances in which the bacteria were observed to be intrahyphal, the host fungal cell wall was often broken, suggesting intrahyphal existence was opportunistic rather than obligatory . The taxonomic position of a bacterium isolated previously from sporocarps exhibiting symptoms similar to those of drippy gill was determined by examining the biochemical and nutritional profiles of the bacterium, and comparing them with other Pseudomonas agarici isolates. J Biol Chem, 2000 Jun 30, 275(26), 20012 - 9 Mechanism of inactivation of ornithine transcarbamoylase by Ndelta -(N'-Sulfodiaminophosphinyl)-L-ornithine, a true transition state analogue? Crystal structure and implications for catalytic mechanism; Langley DB et al.; The crystal structure is reported at 1.8 A resolution of Escherichia coli ornithine transcarbamoylase in complex with the active derivative of phaseolotoxin from Pseudomonas syringae pv . phaseolicola, N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine . Electron density reveals that the complex is not a covalent adduct as previously thought . Kinetic data confirm that N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine exhibits reversible inhibition with a half-life in the order of approximately 22 h and a dissociation constant of K(D) = 1.6 x 10(-12) m at 37 degrees C and pH 8.0 . Observed hydrogen bonding about the chiral tetrahedral phosphorus of the inhibitor is consistent only with the presence of the R enantiomer . A strong interaction is also observed between Arg(57) Nepsilon and the P-N-S bridging nitrogen indicating that imino tautomers of N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine are present in the bound state . An imino tautomer of N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine is structurally analogous to the proposed reaction transition state . Hence, we propose that N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine, with its three unique N-P bonds, represents a true transition state analogue for ornithine transcarbamoylases, consistent with the tight binding kinetics observed. Biochemistry, 2000 Apr 11, 39(14), 3899 - 907 Purification, characterization, and preliminary crystallographic study of copper-containing nitrous oxide reductase from Pseudomonas nautica 617; Prudencio M et al.; The aerobic purification of Pseudomonas nautica 617 nitrous oxide reductase yielded two forms of the enzyme exhibiting different chromatographic behaviors . The protein contains six copper atoms per monomer, arranged in two centers named Cu(A) and Cu(Z) . Cu(Z) could be neither oxidized nor further reduced under our experimental conditions, and exhibits a 4-line EPR spectrum (g(x)=2.015, A(x)=1.5 mT, g(y)=2.071, A(y)=2 mT, g(z)=2.138, A(z)=7 mT) and a strong absorption at approximately 640 nm . Cu(A) can be stabilized in a reduced EPR-silent state and in an oxidized state with a typical 7-line EPR spectrum (g(x)=g(y)= 2.021, A(x) = A(y)=0 mT, g(z) = 2.178, A(z)= 4 mT) and absorption bands at 480, 540, and approximately 800 nm . The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the Cu(A) center . In form A, Cu(A) is predominantly oxidized (S = (1)/(2), Cu(1.5+)-Cu(1.5+)), while in form B it is mostly in the one-electron reduced state (S = 0, Cu(1+)-Cu(1+)) . In both forms, Cu(Z) remains reduced (S = 1/2) . Complete crystallographic data at 2.4 A indicate that Cu(A) is a binuclear site (similar to the site found in cytochrome c oxidase) and Cu(Z) is a novel tetracopper cluster {Brown, K., et al . (2000) Nat . Struct . Biol . (in press)} . The complete amino acid sequence of the enzyme was determined and comparisons made with sequences of other nitrous oxide reductases, emphasizing the coordination of the centers . A 10.3 kDa peptide copurified with both forms of nitrous oxide reductase shows strong homology with proteins of the heat-shock GroES chaperonin family. Microbiology, 2000 Mar, 146 ( Pt 3), 649 - 57 The gene pvaB encodes oxidized polyvinyl alcohol hydrolase of Pseudomonas sp . strain VM15C and forms an operon with the polyvinyl alcohol dehydrogenase gene pvaA; Shimao M et al.; A 5.7 kbp SphI fragment containing the polyvinyl alcohol (PVA) dehydrogenase gene pvaA and its 1.9 kbp 5'-flanking region was cloned from the PVA-degrading bacterium Pseudomonas sp . VM15C . The pvaB gene, encoding oxidized PVA hydrolase, was found in the region upstream of pvaA . Sequence data and expression studies indicated that pvaA and B constitute an operon in the order pvaBA . The pvaB gene encoded a protein of 379 amino acid residues (40610 Da), and a lipoprotein signal sequence and the lipase consensus sequence, Gly-X-Ser-X-Gly, characteristic of the active-site serine region in serine hydrolases, were detected in the deduced amino acid sequence . The pvaB product with the pvaA product constituted an enzyme system for the cleavage of PVA molecules . The pvaA product introduced beta-diketone groups into the PVA molecule, and the pvaB product hydrolysed these beta-diketone groups in oxidized PVA . The pvaB product also hydrolysed 4,6-nonanedione at a low rate, but not acetylacetone or 5-nonanone . It was completely inhibited by PMSF and was concluded to be a serine hydrolase . There were no proteins showing high similarity to the pvaB product in the databases, but minor similarity to a number of serine hydrolases including polyhydroxyalkanoate depolymerases was apparent. Appl Environ Microbiol, 2000 Apr, 66(4), 1416 - 22 Isolation of bacteriophages specific to a fish pathogen, Pseudomonas plecoglossicida, as a candidate for disease control; Park SC et al.; Two types of bacteriophage specific to Pseudomonas plecoglossicida, the causative agent of bacterial hemorrhagic ascites disease in cultured ayu fish (Plecoglossus altivelis), were isolated from diseased ayu and the rearing pond water . One type of phage, which formed small plaques, was tentatively classified as a member of the family Myoviridae, and the other type, which formed large plaques, was classified as a member of the family Podoviridae . All 27 strains of P . plecoglossicida examined, which were isolated from diseased ayu from geographically different areas in 1991 to 1999, exhibited quite similar sensitivities to either type of phage . One strain of P . plecoglossicida was highly virulent for ayu, and the 50% lethal dose (LD(50)) when intramuscular injection was used was 10(1.2) CFU fish(-1); in contrast, phage-resistant variants of this organism were less virulent (LD(50), >10(4) CFU fish(-1)) . Oral administration of phage-impregnated feed to ayu resulted in protection against experimental infection with P . plecoglossicida . After oral administration of P . plecoglossicida cells of this bacterium were always detected in the kidneys of control fish that did not receive the phage treatment, while the cells quickly disappeared from the phage-treated fish . Bacterial growth in freshwater was lower in the presence of phage, and the number of phage PFU increased rapidly . These results suggest that it may be possible to use phage to control the disease caused by P . plecoglossicida. Appl Environ Microbiol, 2000 Apr, 66(4), 1385 - 92 Poly(3-hydroxyvalerate) depolymerase of Pseudomonas lemoignei; Schober U et al.; Pseudomonas lemoignei is equipped with at least five polyhydroxyalkanoate (PHA) depolymerase structural genes (phaZ1 to phaZ5) which enable the bacterium to utilize extracellular poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV), and related polyesters consisting of short-chain-length hxdroxyalkanoates (PHA(SCL)) as the sole sources of carbon and energy . Four genes (phaZ1, phaZ2, phaZ3, and phaZ5) encode PHB depolymerases C, B, D, and A, respectively . It was speculated that the remaining gene, phaZ4, encodes the PHV depolymerase (D . Jendrossek, A . Frisse, A . Behrends, M . Andermann, H . D . Kratzin, T . Stanislawski, and H . G . Schlegel, J . Bacteriol . 177:596-607, 1995) . However, in this study, we show that phaZ4 codes for another PHB depolymeraes (i) by disagreement of 5 out of 41 amino acids that had been determined by Edman degradation of the PHV depolymerase and of four endoproteinase GluC-generated internal peptides with the DNA-deduced sequence of phaZ4, (ii) by the lack of immunological reaction of purified recombinant PhaZ4 with PHV depolymerase-specific antibodies, and (iii) by the low activity of the PhaZ4 depolymerase with PHV as a substrate . The true PHV depolymerase-encoding structural gene, phaZ6, was identified by screening a genomic library of P . lemoignei in Escherichia coli for clearing zone formation on PHV agar . The DNA sequence of phaZ6 contained all 41 amino acids of the GluC-generated peptide fragments of the PHV depolymerase . PhaZ6 was expressed and purified from recombinant E . coli and showed immunological identity to the wild-type PHV depolymerase and had high specific activities with PHB and PHV as substrates . To our knowledge, this is the first report on a PHA(SCL) depolymerase gene that is expressed during growth on PHV or odd-numbered carbon sources and that encodes a protein with high PHV depolymerase activity . Amino acid analysis revealed that PhaZ6 (relative molecular mass {M(r)}, 43,610 Da) resembles precursors of other extracellular PHA(SCL) depolymerases (28 to 50% identical amino acids) . The mature protein (M(r), 41,048) is composed of (i) a large catalytic domain including a catalytic triad of S(136), D(211), and H(269) similar to serine hydrolases; (ii) a linker region highly enriched in threonine residues and other amino acids with hydroxylated or small side chains (Thr-rich region); and (iii) a C-terminal domain similar in sequence to the substrate-binding domain of PHA(SCL) depolymerases . Differences in the codon usage of phaZ6 for some codons from the average codon usage of P . lemoignei indicated that phaZ6 might be derived from other organisms by gene transfer . Multialignment of separate domains of bacterial PHA(SCL) depolymerases suggested that not only complete depolymerase genes but also individual domains might have been exchanged between bacteria during evolution of PHA(SCL) depolymerases. J Biochem (Tokyo), 2000 Apr, 127(4), 597 - 601 Glutathione as an essential factor for chaperon-mediated activation of lactonizing lipase (LipL) from Pseudomonas sp . 109; Tanaka J et al.; Pseudomonas sp . 109 produces a unique lipase (LipL) which efficiently catalyzes intramolecular transesterification of omega-hydroxyesters to form macrocyclic lactones . The production of the enzymatically active LipL requires a specific molecular chaperon (LimL protein) together with a low-M(r) lipase-activation-factor (LAF) of unknown structure . From 50 g of Pseudomonas cells, 2.15 mg of LAF was purified as a sulfobenzofurazanyl derivative after methanol extraction, derivatization, and C(18) reverse-phase HPLC . One-dimensional and two-dimensional 600 MHz (1)H-NMR and fast atom bombardment mass spectrometry (FAB-MS) revealed that LAF is glutathione . Because several SH compounds (L-cysteine and mercaptoethanol) were similarly effective to native LAF in the activation of LipL, and because only LipL contains two cysteinyl residues forming an intramolecular disulfide bond, it is concluded that the reduction of and reformation of the intramolecular disulfide bond of LipL is essential to liberate free and fully active LipL. Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 458 - 9 Crystallization and preliminary X-ray analysis of cephalosporin C acylase from Pseudomonas sp . strain N176; Kinoshita T et al.; Cephalosporin C acylase from Pseudomonas sp . strain N176, a heterodimer of 25 and 58 kDa, has been crystallized using polyethylene glycol 6000 as precipitant . The crystals are orthorhombic and have unit-cell parameters a = 141.41, b = 192.10, c = 80.75 A . They belong to space group P2(1)2(1)2(1) and diffract to at least 2.7 A resolution . Calculations indicate that there are two heterodimers in the asymmetric unit . The structure is being solved by molecular replacement using penicillin G acylase from Escherichia coli as a search model and by multiple isomorphous replacement. Int J Cancer, 2000 Apr 15, 86(2), 269 - 75 Recombinant antibody toxins specific for ErbB2 and EGF receptor inhibit the in vitro growth of human head and neck cancer cells and cause rapid tumor regression in vivo; Azemar M et al.; Overexpression of the ErbB2 and epidermal growth factor receptor (EGFR) tyrosine kinases is frequently observed in squamous cell carcinomas of the head and neck, and has been correlated with shorter overall survival . By immunoblot analysis, we have found EGFR and ErbB2 expression in 6 out of 6 established head and neck cancer cell lines . Elevated EGFR protein levels were noted in 3 and elevated ErbB2 levels in 5 of them . Significant expression of EGFR and ErbB2 was also detected in 17 of 47 and 26 of 45 primary tumor samples . Due to their enhanced expression on the tumor cell surface, these receptors can be regarded as suitable targets for directed cancer therapy . We have analyzed the antitumoral activity of recombinant single-chain antibody toxins specific for ErbB2 and EGFR against head and neck cancer cells in vitro and in vivo . The recombinant toxins consist of the variable domains of the heavy and light chains of monoclonal antibodies (MAbs) genetically fused to a truncated Pseudomonas exotoxin A (ETA) . At low concentrations, the ErbB2-specific single-chain antibody (scFv) toxin scFv(FRP5)-ETA and the EGFR-specific toxins scFv(225)-ETA and scFv(14E1)-ETA inhibited the in vitro growth of established head and neck cancer cell lines and primary tumor cells . In a nude mouse tumor model, intratumoral injection of the antibody toxins resulted in the rapid regression of subcutaneously growing CAL 27 tumor xenografts, with scFv(FRP5)-ETA and scFv(14E1)-ETA treatment being most effective and leading to the cure of up to 50% of the animals . Our results suggest that EGFR and ErbB2-specific antibody toxins may become valuable therapeutic reagents for the treatment of squamous cell carcinomas of the head and neck . Biosci Biotechnol Biochem, 2000 Feb, 64(2), 299 - 305 1-aminocyclopropane-1-carboxylate (ACC) deaminase induced by ACC synthesized and accumulated in Penicillium citrinum intracellular spaces; Jia YJ et al.; We have already described how 1-aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of the plant hormone ethylene, is synthesized in Penicillium citrinum through the same reaction by the catalysis of ACC synthase {EC 4.4.1.14} as in higher plants . In addition, ACC deaminase {EC 4.1.99.4}, which degrades ACC to 2-oxobutyrate and ammonia, was also purified from this strain . To study control of induction of ACC deaminase in this organism, we have isolated and analyzed the cDNA of P . citrinum ACC deaminase and studied the expression of ACC deaminase mRNA in P . citrinum cells . By the analysis of peptides from the digests of the purified and modified ACC deaminase with lysylendopeptidase, 70 % of its amino acid sequences were obtained . These amino acid sequences were used to identify a cDNA, consisting of 1,233 bp with an open reading frame of 1,080 bp encoding ACC deaminase with 360 amino acids . The deduced amino acids from the cDNA are identical by 52% and 45% to those of enzymes of Pseudomonas sp . ACP and Hansenula saturnus . Through Northern blot analysis, we found that the mRNA of ACC deaminase was expressed in P . citrinum cells grown in a medium containing 0.05% L-methionine . These findings suggest that ACC synthesized by ACC synthase and accumulated in P . citrinum intracellular spaces can induce the ACC deaminase that degrades the ACC. Acta Microbiol Immunol Hung, 2000, 47(1), 63 - 73 Some evidences for the involvement of plasmid in diuron herbicide degradation; El-Deeb BA et al.; Pseudomonas sp . strain Bk8 was isolated from field soil contaminated with different urea-herbicides . This strain is a plasmid (pBkB)-harbouring organism capable of complete degradation of diuron herbicide . Plasmid-cured strain Bk8M was obtained by treatment of Pseudomonas sp . Bk8 with Mitomycin C . This cured strain is capable of only partial degradation of diuron side chain and accumulated a phenolic compound in the medium during growing on diuron as a sole source of carbon and energy . Conjugation experiment was carried out using Bk8M as a recipient and Bk8 as a donor of pBk8 plasmid . The transconjugant was able to degrade a diuron without accumulation of phenolic compound . It was proposed that plasmid pBk8 is self-transmissible and involved in the degradation of diuron aromatic ring but it is not connected with the transformation of diuron into diuron phenol compound. Genetika, 2000 Jan, 36(1), 28 - 33 {Constructing of system of genetic analysis in Pseudomonas mendocina}; Vasilenko SL et al.; A Tn10-containing variant of the pRK2013 plasmid, pRK2013-7, was used in the genetic analysis of Pseudomonas mendocina as chromosome-mobilizing inheritable factor that is able to integrate into the bacterial chromosome and transfer genetic markers with a frequency ranging from 3.2 x 10(-7) to 3.5 x 10(-3) . The results of interrupted matings allowed localization of 10 genetic markers . This system of genetic analysis is suitable for P . mendocina mapping. FEMS Microbiol Lett, 2000 Apr 1, 185(1), 23 - 7 Cloning and overexpression of a tyrosinase gene mel from Pseudomonas maltophila; Wang G et al.; The tyrosinase gene (mel), which is responsible for melanin formation, was isolated by shotgun cloning of SalI fragments of Pseudomonas maltophila DNA . A 0.7-kb SalI fragment in the recombinant plasmid pWSY8 imparted the ability to synthesize melanin to an Escherichia coli host HB101 . The nucleotide sequence of this DNA fragment revealed an open reading frame of 504 bp, encoding a protein of 169 amino acids . The fragment containing the mel gene was then cloned into an expression plasmid pPAS1 under the control of a promoter isolated from the host, P . maltophilia AT18 . This strain increased the melanin production by 70.6% compared with the strain HB101/pWSY8, in which the cloned mel gene was under the control of the lac promoter from the vector pUC18. Br J Dermatol, 2000 Feb, 142(2), 321 - 30 Development of chimeric molecules for recognition and targeting of antigen-specific B cells in pemphigus vulgaris; Proby CM et al.; Pemphigus vulgaris (PV) is an autoimmune blistering disease characterized by circulating pathogenic IgG antibodies against desmoglein 3 (Dsg3) . The purpose of this study was to develop chimeric molecules for specific recognition and elimination of autoimmune B cells in PV . Mouse hybridoma cell lines producing anti-Dsg3 antibody (5H10, 12A2) were developed as an in vitro model system for targeting B cells . Dsg3-GFP, a baculoprotein containing the entire extracellular domain of Dsg3 fused with green fluorescence protein, recognized and targeted the hybridoma cells through their surface immunoglobulin receptors in an antigen-specific way . The epitopes of these monoclonal antibodies were mapped on the amino terminal EC1 and part of EC2, a region considered functionally important in cadherins . Chimeric toxin molecules containing the mapped region (Dsg3deltaN1) and modified Pseudomonas exotoxin were produced in bacteria (Dsg3deltaN1-PE40-KDEL, PE3 7-Dsg3deltaN1-KDEL) and tested in vitro on hybridoma cell lines . The chimeric toxins, but not Dsg3deltaN1 alone, showed dose-dependent toxic activity with a reduction in hybridoma cell number to 40-60% of toxin-negative control cultures, compared with little or no effect on anti-Dsg3-negative hybridoma cells . Furthermore, these toxins showed toxic effects on anti-Dsg3 IgG-producing B cells from Dsg3deltaN1-immunized mice, with a 60% reduction in cell number compared with Dsg3deltaN1 alone . Thus, specific recognition and targeting of antigen-specific B cells in PV was demonstrated; this strategy may hold promise as a future therapeutic option for PV and other autoimmune diseases. Eur J Biochem, 2000 Apr, 267(7), 1957 - 65 Expression, stability and performance of the three-component alkane mono-oxygenase of Pseudomonas oleovorans in Escherichia coli; Staijen IE et al.; We tested the synthesis and in vivo function of the inducible alkane hydroxylase of Pseudomonas oleovorans GPo1 in several Escherichia coli recombinants . The enzyme components (AlkB, AlkG and AlkT) were synthesized at various rates in different E . coli hosts, which after induction produced between twofold and tenfold more of the Alk components than did P . oleovorans . The enzyme components were less stable in recombinant E . coli hosts than in P . oleovorans . In addition, the specific activity of the alkane mono-oxygenase component AlkB was five or six times lower in E . coli than in P . oleovorans . Evidently, optimal functioning of the hydroxylase system requires factors or a molecular environment that are available in Pseudomonas but not in E . coli . These factors are likely to include correct interactions of AlkB with the membrane and incorporation of iron into the AlkG and AlkB apoproteins. Biochemistry, 2000 Mar 28, 39(12), 3351 - 9 Catalytic activity of the D38A mutant of 3-oxo-Delta 5-steroid isomerase: recruitment of aspartate-99 as the base; Henot F et al.; 3-oxo-Delta(5)-steroid isomerase (KSI) from Comamonas (Pseudomonas) testosteroni catalyzes the isomerization of beta,gamma-unsaturated 3-oxosteroids to their conjugated isomers through an intermediate dienolate . Residue Asp-38 (pK(a) 4.57) acts as a base to abstract a proton from C-4 of the substrate to form an intermediate dienolate, which is then reprotonated on C-6 . Both Tyr-14 (pK(a) 11.6) and Asp-99 (pK(a) >/= 9.5) function as hydrogen-bond donors to O-3 of the steroid, helping to stabilize the transition states . Mutation of the active-site base Asp-38 to the weakly basic Asn (D38N) has previously been shown to result in a >10(8)-fold decrease of catalytic activity . In this work, we describe the preparation and kinetic analysis of the Ala-38 (D38A) mutant . Unexpectedly, D38A has a catalytic turnover number (k(cat)) that is ca . 10(6)-fold greater than the value for D38N and only about 140-fold less than that for wild type . Kinetic studies as a function of pH show that D38A-catalyzed isomerization involves two groups, with pK(a) values of 4.2 and 10.4, respectively, in the free enzyme, which are assigned to Asp-99 and either Tyr-14 or Tyr-55 . A mechanism for D38A is proposed in which Asp-99 is recruited as the catalytic base, with stabilization of the intermediate dienolate ion and the flanking transition states provided by hydrogen bonding from both Tyr-14 and Tyr-55 . This mechanism is supported by the lack of detectable activity of the D38A/D99N, D38A/Y14F, and D38A/Y55F double mutants. Biol Pharm Bull, 2000 Mar, 23(3), 279 - 82 Role of MerT and MerP from Pseudomonas K-62 plasmid pMR26 in the transport of phenylmercury; Kiyono M et al.; To investigate the individual role of MerT and MerP encoded by Pseudomonas K-62 pMR26 in the transport of phenylmercury, a series of mutants with a specific point mutation in merT and/or genetic deletion in merP were constructed and transformed into Escherichia coli XL1-Blue . Transport of phenylmercury across the cytoplasmic membrane of E . coli mediated by MerT and MerP was inhibited by NaCN and by cold temperatures . Deletion of merP reduced, but did not completely abolish the C6H5Hg+-hyperuptake and -hypersensitive phenotypes suggesting that transport of phenylmercury into the cytoplasm of E . coli is still occurring . Mutations of the vicinal cysteine residues (Cys24 and Cys25) in the first transmembrane region of MerT to serine caused complete loss of Hg2+-hyperuptake and -hypersensitivity, whereas the mutations did not affect the C6H5Hg+-hyperuptake and -hypersensitive phenotypes . In addition, no additive effect on the C6H5Hg+-hyperuptake and -hypersensitive phenotypes was found, when mutations of the two cysteines in MerT to serine were further introduced in the merP-deleted mutants . These results clearly demonstrated that the vicinal cysteine residues of MerT are not involved in the transport of C6H5Hg+, but indeed are involved in the transport of Hg2+ as previously reported. Plant Sci, 2000 May 15, 154(1), 71 - 81 Molecular cloning of a soybean class III beta-1,3-glucanase gene that is regulated both developmentally and in response to pathogen infection; Cheong YH et al.; We isolated and characterized a soybean gene (SGN1) encoding a basic beta-1,3-glucanase that is a plant class III isoform of beta-1,3-glucanase . The deduced amino acid sequence of the SGN1 gene is similar to that of the PR-Q'b gene, the basic class III beta-1,3-glucanase of tomato . Based on RNA blot hybridization, SGN1 gene expression was detected in all tissues of 4-day old seedlings, but it was present only in root tissue of 30-day old plants . GUS expression analysis carried out in transgenic tobacco plants harboring a SGN1::GUS reporter gene revealed the same expression pattern . Furthermore, the expression of SGN1 was strongly induced by a variety of defense-related signals, such as treatment with H(2)O(2), wounding, or treatment with fungal elicitor prepared from Phytophthora spp as well as inoculation with Pseudomonas syringae . However, the expression level of SGN1 was hardly induced with jasmonate, ethephon and salicylate . Overall the results suggest that the SGN1 may play a role in both plant development and plant defense against pathogen attack. Acta Crystallogr D Biol Crystallogr, 2000 Mar, 56 ( Pt 3), 313 - 21 Pseudo-symmetry characterization and refinement of a trigonal crystal form of naphthalene 1,2-dioxygenase; Carredano E et al.; Two trigonal crystal structures of naphthalene 1,2-dioxygenase from Pseudomonas sp . NCIB 9816-4 have been refined at 2.6 A resolution . The space group is R3, with four heterodimers in the asymmetric unit . The crystallographic threefold axis coincides with the symmetry axis of the active molecule, a mushroom-shaped alpha(3)beta(3) hexamer . The crystal is formed by symmetrical contacts between the hexamers on three different interaction surfaces, one on the beta-subunit and the other two on the alpha--subunits . Nickel ions mediate one of the alpha-subunit interactions . The two other types of packing contacts sustain two interlaced and almost independent crystal patterns with significantly different temperature factors . The space group of the individual crystal patterns is R32, with the corresponding twofold axes parallel to each other . The interactions between the crystal patterns separate the two parallel twofolds, eliminating the twofold symmetry for the whole crystal . The differences in temperature factors among the molecules in the asymmetric unit have been refined and are different for the two refined structures . An analysis of the structure factors of the pseudo-equivalent reflections showed that their differences lie in their phases and not in their amplitudes, suggesting that R(merge) is not an appropriate indicator for revealing the correct point group. J Biol Chem, 2000 Mar 17, 275(11), 7566 - 73 Recombinant toxins that bind to the urokinase receptor are cytotoxic without requiring binding to the alpha(2)-macroglobulin receptor; Rajagopal V et al.; The alpha(2-)macroglobulin receptor (alpha(2)MR) has been reported to mediate the internalization of the urokinase plasminogen activator receptor (uPAR) via ligand binding to both receptors . To target malignant uPAR-expressing cells and to determine whether uPAR can internalize without ligand binding to alpha(2)MR, we engineered two recombinant toxins, ATF-PE38 and ATF-PE38KDEL . Each consists of the amino-terminal fragment (ATF) of human urokinase and a truncated form of Pseudomonas exotoxin (PE) devoid of domain Ia, which binds alpha(2)MR . ATF-PE38 and ATF-PE38KDEL were cytotoxic toward malignant uPAR-bearing cells, with IC(50) values as low as 0.02 ng/ml (0.3 pM) . Cytotoxicity could be blocked using either recombinant urokinase or free ATF, indicating that the cytotoxicity of the recombinant toxins was specific . Radiolabeled ATF-PE38 had high affinity for uPAR (K(d) = 0.4-8 nM) on a variety of different malignant cell types and internalized at a rate similar to that of ATF . The cytotoxicity was not diminished by receptor-associated protein, which binds and shields the alpha(2)MR from other proteins, or by incubation with phorbol myristate acetate, which is known to decrease the number of alpha(2)MRs in U937 cells or by antibodies to alpha(2)MR . Therefore, these recombinant toxins appear to internalize via uPAR without association with the alpha(2)MR. Appl Microbiol Biotechnol, 2000 Feb, 53(2), 167 - 72 Production of polyhydroxyalkanoic acids by Ralstonia eutropha and Pseudomonas oleovorans from an oil remaining from biotechnological rhamnose production; Fuchtenbusch B et al.; Screening experiments identified several bacteria which were able to use residual oil from biotechnological rhamnose production as a carbon source for growth . Ralstonia eutropha H16 and Pseudomonas oleovorans were able to use this waste material as the sole carbon source for growth and for the accumulation of polyhydroxyalkanoic acids (PHA) . R . eutropha and P . oleovorans accumulated PHA amounting to 41.3% and 38.9%, respectively, of the cell dry mass, when these strains were cultivated in mineral salt medium with the oil from the rhamnose production as the sole carbon source . The accumulated PHA isolated from R . eutropha consisted of only 3-hydroxybutyric acid, whereas the PHA isolated from P . oleovorans consisted of 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid, and 3-hydroxydodecanoic acid . The composition was confirmed by gas chromatography of the isolated polyesters . Batch and fed-batch cultivations in stirred-tank reactors were done. Ann Oncol, 2000, 11 Suppl 1, 101 - 6 T-cell receptors for cytokines: targets for immunotherapy of leukemia/lymphoma; Waldmann TA; BACKGROUND: Cytokine receptors are exceptionally valuable targets for immunotherapy . For example, the high affinity IL-2 receptor is expressed by abnormal T cells in patients with certain lymphoid malignancies or autoimmune disorders and in individuals rejecting allografts whereas it is not expressed by normal resting cells . DESIGN: To exploit this difference in receptor expression in normal resting cells and leukemic cells we have introduced different forms of IL-2 receptor directed therapy including an unmodified murine antibody to the alpha subunit of the IL-2 receptor (anti-Tac), humanized anti-Tac as well as this antibody armed with truncated Pseudomonas exotoxin or alpha- and beta-emitting radionuclides (e.g., 211At and 90Y) . In particular, unmodified murine anti-Tac was used in the therapy of HTLV-I-associated adult T-cell leukemia (ATL) . RESULTS: Six of nineteen patients treated with this antibody underwent a partial (four) or complete (two) remission . In a subsequent clinical trial involving anti-Tac armed with 90Y over 50% of the patients with ATL treated underwent a partial or complete remission . CONCLUSIONS: New agents under development include humanized antibodies directed toward shared cytokine receptors such as IL-2/15R beta used by both IL-2 and IL-15 as well as to a shared signal transduction element Jak3 utilized by the T-cell stimulatory cytokines IL-2, IL-4, IL-7, IL-9 and IL-15 . Thus our emerging understanding of cytokine receptors and their signaling pathways taken in conjunction with the ability to produce humanized antibodies armed with radionuclides or toxins are providing novel perspectives for the treatment of leukemia and lymphoma. Mol Plant Microbe Interact, 2000 Mar, 13(3), 342 - 6 Syringolin-mediated activation of the Pir7b esterase gene in rice cells is suppressed by phosphatase inhibitors; Hassa P et al.; Inoculation of rice plants (Oryza sativa) with the nonhost pathogen Pseudomonas syringae pv . syringae leads to the activation of defense-related genes and ultimately to induced resistance against the rice blast fungus Pyricularia oryzae . One of the molecular determinants of P . syringae pv . syringae that is recognized by the plant cells and evokes these defense responses is syringolin A, an elicitor that is secreted by the bacteria under appropriate conditions . In order to investigate signal transduction events elicited by syringolin A, the response of cultured rice cells to syringolin A application was analyzed . Cultured rice cells were able to sense syringolin A at concentrations in the nanomolar range as observed by the transient accumulation of Pir7b esterase transcripts . Syring