Planta, 2002 Jun, 215(2), 239 - 47 Epub 2002 Mar 22. Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance; Li L et al.; Polyphenol oxidases (PPOs; EC 1.10.3.2 or EC 1.14.18.1) catalyzing the oxygen-dependent oxidation of phenols to quinones are ubiquitous among angiosperms and assumed to be involved in plant defense against pests and pathogens . In order to investigate the role of PPO in plant disease resistance, we made transgenic tomato ( Lycopersicon esculentum Mill . cv . Money Maker) plants that overexpressed a potato ( Solanum tuberosum L.) PPO cDNA under control of the cauliflower mosaic virus 35S promoter . The transgenic plants expressed up to 30-fold increases in PPO transcripts and 5- to 10-fold increases in PPO activity and immunodetectable PPO . As expected, these PPO-overexpressing transgenic plants oxidized the endogenous phenolic substrate pool at a higher rate than control plants . Three independent transgenic lines were selected to assess their interaction with the bacterial pathogen Pseudomonas syringae pv . tomato . The PPO-overexpressing tomato plants exhibited a great increase in resistance to P . syringae . Compared with control plants, these transgenic lines showed less severity of disease symptoms, with over 15-fold fewer lesions, and strong inhibition of bacterial growth, with over 100-fold reduction of bacterial population in the infected leaves . These results demonstrate the importance of PPO-mediated phenolic oxidation in restricting plant disease development. Plant J, 2002 May, 30(4), 467 - 80 The Arabidopsis hrl1 mutation reveals novel overlapping roles for salicylic acid, jasmonic acid and ethylene signalling in cell death and defence against pathogens; Devadas SK et al.; Defence against pathogens in Arabidopsis is orchestrated by at least three signalling molecules: salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) . The hrl1 (hypersensitive response-like lesions 1) mutant of Arabidopsis is characterized by spontaneous necrotic lesions, accumulation of reactive oxygen species, constitutive expression of SA- and ET/JA-responsive defence genes, and enhanced resistance to virulent bacterial and oomycete pathogens . Epistasis analyses of hrl1 with npr1, etr1, coi1 and SA-depleted nahG plants revealed novel interactions between SA and ET/JA signalling pathways in regulating defence gene expression and cell death . RNA gel-blot analysis of RNA isolated separately from the lesion+ and the lesion- leaves of double mutants of hrl1 revealed different signalling requirements for the expression of defence genes in these tissues . Expression of the ET/JA-responsive PDF1.2 gene was markedly reduced in hrl1 npr1 and in SA-depleted hrl1 nahG plants . In hrl1 nahG plants, expression of PDF1.2 was regulated by benzathiadiazole in a concentration-dependent manner: induced at low concentration and suppressed at high concentration . The hrl1 etr1 plants lacked systemic PR-1 expression, and exhibited compromised resistance to virulent Pseudomonas syringae and Peronospora parasitica . Inhibiting JA responses in hrl1 coi1 plants lead to exaggerated cell death and severe stunting of plants . Finally, the hrl1 mutation lead to elevated expression of AtrbohD, which encodes a major subunit of the NADPH oxidase complex . Our results indicate that defence gene expression and resistance against pathogens in hrl1 is regulated synergistically by SA and ET/JA defence pathways. Eur J Biochem, 2002 May, 269(10), 2498 - 505 Structural determination of lipid A of the lipopolysaccharide from Pseudomonas reactans . A pathogen of cultivated mushrooms; Silipo A et al.; The chemical structure of lipid A from the lipopolysaccharide of the mushroom-associated bacterium Pseudomonas reactans, a pathogen of cultivated mushroom, was elucidated by compositional analysis and spectroscopic methods (MALDI-TOF and two-dimensional NMR) . The sugar backbone was composed of the beta-(1'-->6)-linked d-glucosamine disaccharide 1-phosphate . The lipid A fraction showed remarkable heterogeneity with respect to the fatty acid and phosphate composition . The major species are hexacylated and pentacylated lipid A, bearing the (R)-3-hydroxydodecanoic acid {C12:0 (3OH)} in amide linkage and a (R)-3-hydroxydecanoic {C10:0 (3OH)} in ester linkage while the secondary fatty acids are present as C12:0 and/or C12:0 (2-OH) . A nonstoichiometric phosphate substitution at position C-4' of the distal 2-deoxy-2-amino-glucose was detected . Interestingly, the pentacyl lipid A is lacking a primary fatty acid, namely the C10:0 (3-OH) at position C-3' . The potential biological meaning of this peculiar lipid A is also discussed. Mikrobiologiia, 2002 Mar-Apr, 71(2), 240 - 6 {Some characteristics of Pseudomonas syringae pv . maculicola dissociants}; Iakovleva LM et al.; Pseudomonas syringae pv . maculicola dissociants producing colonies of different morphotype were found to possess similar biochemical and serological properties but different virulence to the host plant . The heterogeneous extracellular and intracellular lipopolysaccharide-protein complexes of the dissociants differed in their chemical composition and biological activity towards test plants. Cancer Res, 2002 May 15, 62(10), 2848 - 55 A recombinant CD7-specific single-chain immunotoxin is a potent inducer of apoptosis in acute leukemic T cells; Peipp M et al.; A recombinant immunotoxin was constructed from the hybridoma antibody TH-69 directed against human CD7, a surface antigen of leukemic T cells . The antibody was subcloned as a single chain Fv (scFv) fragment and genetically linked to a truncated Pseudomonas exotoxin A fragment containing the catalytic domains II and III but lacking the receptor binding domain I . Domain I was replaced by the scFv, thus conferring restricted specificity for CD7-positive cells . The bacterially expressed and purified toxin retained binding specificity for CD7-positive cells . It promoted apoptosis in two CD7-positive cell lines derived from T-lineage acute lymphoblastic leukemias, CEM and Jurkat, but not in the CD7-negative B-lymphoid lines REH, Nalm-6, and SEM . Maximum killing in excess of 95% was reached after 96 h in CEM and Jurkat cells with a single dose of 100 ng/ml . Cells treated with a similarly constructed scFv-exotoxin A immunotoxin against melanoma-associated chondroitin sulfate proteoglycan, an antigen absent from leukemic T cells, remained unaffected . Lysis of target cells occurred via apoptosis as evidenced by staining with Annexin V and specific cleavage of poly(ADP-ribose) polymerase . Approximately 20% of leukemic cells from a patient with CD7-positive acute T-cell leukemia kept in long-term primary culture for 30 cell generations were killed within 96 h after treatment with the toxin . These findings justify further evaluation of the agent in view of potential therapeutic applications. Mol Cells, 2002 Apr 30, 13(2), 309 - 14 DNA markers for identification of Pseudomonas syringae pv . actinidiae; Koh YJ et al.; The specific DNA fragment was screened by RAPD analysis of Pseudomonas syringae pv . actinidiae, as well as similar strains that were isolated from kiwifruits . The primer C24 detected a fragment that is specific in P . syringae pv . actinidiae . This fragment was cloned . The pathovar-specific fragment was detected from a Southern blot analysis of the genomic DNAs of P . syringae pv . actinidiae using the cloned fragment as a probe . The sequence size of the cloned fragment was determined as 675 bp . A DNA Database search suggested that the fragment was a novel one . Approximately 9 kb of a single fragment was detected only in the P . syringae pv . actinidiae by a Southern blot analysis of the genomic DNAs of P . syringae pv . actinidiae . Similar strains were also detected with the use of the cloned fragment as a probe . Since the genomic DNAs were digested with HindIII without a cleavage site, the result reveals that the cloned fragment exists on the genome of P . syringae pv . actinidiae as a single copy . A pair of primers that produced a 492 bp single fragment (only in the strains of P . syringae pv . actinidiae) were synthesized, based on the pathovar-specific sequences of the cloned fragment of P . syringae pv . actinidiae . The development of the primers and probe made it possible to diagnose the bacterial canker infection from leaves or trunks of kiwifruit trees before any symptom appeared on the tree. Int J Colorectal Dis, 2002 Mar, 17(2), 77 - 84 The in vitro anti-inflammatory effects of recombinant anti-CD25 immunotoxin on lamina propria T cells of patients with inflammatory bowel disease are not sufficient to cure experimental colitis in mice; Pfister K et al.; BACKGROUND AND AIMS: In chronic inflammatory bowel disease (IBD) such as Crohn's disease and ulcerative colitis an aberrant mucosal immune regulation is observed accompanied by upregulation of proinflammatory cytokines . Lamina propria T cells of inflamed mucosa have an activated phenotype characterized by increased expression of surface markers such as CD25 . We therefore determined the anti-inflammatory effect of a recombinant immunotoxin consisting of an anti-CD25 single chain variable fragment (scFv) fused to a deletion mutant of Pseudomonas exotoxin A {RFT5(scFv)ETA'} on isolated lamina propria lymphocytes of patients with IBD and in the murine model of trinitrobenzene sulfonic acid (TNBS) induced colitis . PATIENTS AND/METHODS: Lamina propria lymphocytes of 25 patients with IBD and 19 control patients were stimulated in absence or presence of RFT5(scFv)ETA' . Interferon-gamma production was determined in the supernatant by ELISA and the induction of apoptosis by flow cytometry after propidium iodide staining . BALB/c mice received TNBS intrarectally and were treated with RFT5(scFv)ETA' . RESULTS: In vitro the administration of RFT5(scFv)ETA' significantly reduced interferon-gamma production and increased apoptosis in lamina propria lymphocytes isolated of inflamed mucosa . However, this contrainflammatory regulation did not result in gain of weight or increased life span in experimental colitis in vivo . CONCLUSION: In addition to the downregulation of the proinflammatory cytokine in vitro, RFT5(scFv)ETA' induced neither a direct nor a bystander effect in an in vivo model of colitis . Therefore our data do not support potential therapeutic implications of targeting CD25 by RFT5(scFv)ETA' in chronic IBD. Biochim Biophys Acta, 2002 May 20, 1597(1), 60 - 6 Fluorescent inhibitors reveal solvent-dependent micropolarity in the lipid binding sites of lipases; Oskolkova OV et al.; Triacylglycerol analogue p-nitrophenyl phosphonates specifically react with the active-site serine of lipolytic enzymes to give covalent lipase-inhibitor complexes, mimicking the first transition state which is involved in lipase-mediated ester hydrolysis . Here we report on a new type of phosphonate inhibitors containing a polarity-sensitive fluorophore to monitor micropolarity around the active site of the enzyme in different solvents . The respective compounds are hexyl and methyl dimethylamino-naphthalenecarbonylethylmercaptoethoxy-phosphonates . The hexyl phosphonate derivative was reacted with lipases from Rhizopus oryzae (ROL), Chromobacterium viscosum (CVL), and Pseudomonas cepacia (PCL) . The resulting lipid-protein complexes were characterized in solution with respect to water penetration into the lipid binding site and the associated conformational changes of the proteins as a consequence of solvent polarity changes . We found that the accessibility of the lipid-binding site in all lipases studied was lowest in water . It was much higher when the protein was dissolved in aqueous ethanol . These biophysical effects may contribute to the previously observed dramatic changes of enzyme functions such as activity and stereoselectivity depending on the respective solvents. Biomacromolecules, 2002 May-Jun, 3(3), 525 - 30 Enzymatic degradation of block copolymers prepared from epsilon-caprolactone and poly(ethylene glycol); Li S et al.; Block copolymers were prepared by ring-opening polymerization of epsilon-caprolactone in the presence of monohydroxyl or dihydroxyl poly(ethylene glycol) (PEG), using Zn powder as catalyst . The resulting poly(epsilon-caprolactone) (PCL)-PEG diblock and PCL-PEG-PCL triblock copolymers were characterized by various analytical techniques such as NMR, size-exclusion chromatography, differential scanning calorimetry, and X-ray diffraction . Both copolymers were semicrystalline polymers, the crystalline structure being of the PCL type . Films were prepared by casting dichloromethane solutions of the polymers on a glass plate . Square samples with dimensions of 10 x 10 mm were allowed to degrade in a pH = 7.0 phosphate buffer solution containing Pseudomonas lipase . Data showed that the introduction of PEG blocks did not decrease the degradation rate of poly(epsilon-caprolactone). Biosci Biotechnol Biochem, 2002 Mar, 66(3), 543 - 8 Purification and characterization of pyridoxal 4-dehydrogenase from Aureobacterium luteolum; Trongpanich Y et al.; A pyridoxal dehydrogenase was purified to homogeneity from Aureobacterium luteolum, which can use pyridoxine as a carbon and nitrogen source, and characterized . The enzyme was a dimeric protein with a subunit molecular weight of 38,000 . It had several properties distinct from those of the partially purified enzyme from Pseudomonas MA-1 . The optimum pH (8.0-8.5) was 0.8-1.3 lower than that of the Pseudomonas enzyme . The Aureobacterium enzyme showed much higher and lower affinities for NAD+ (Km, 0.140 +/- 0.008 mM) and pyridoxal (0.473 +/- 0.109 mM), respectively, than those of the Pseudomonas enzyme . The Aureobacterium enzyme could use NADP+ as a substrate: the reactivity was 6.5% of NAD+ . The enzyme was much more tolerant to metal-chelating agents . Irreversibility of the enzymatic reaction was shared by the two enzymes . No aldehyde dehydrogenase showed similarity to the amino-terminal amino acid sequence of the enzyme. J Bacteriol, 2002 Jun, 184(11), 3146 - 9 Methylation of inorganic and organic selenium by the bacterial thiopurine methyltransferase; Ranjard L et al.; Escherichia coli cells expressing the tpm gene encoding the bacterial thiopurine methyltransferase (bTPMT) are shown to methylate selenite and (methyl)selenocysteine into dimethylselenide (DMSe) and dimethyldiselenide (DMDSe) . E . coli cells expressing tpm from a gene library cosmid clone (harboring a Pseudomonas syringae insert of about 20 kb) also methylated selenate into DMSe and DMDSe . bTPMT is the first methyltransferase shown to be involved in the methylation of these selenium derivatives. J Bacteriol, 2002 Jun, 184(11), 2914 - 24 Identification and physical characterization of the HbpR binding sites of the hbpC and hbpD promoters; Tropel D et al.; Pseudomonas azelaica HBP1 can use 2-hydroxybiphenyl (2-HBP) and 2,2'-dihydroxybiphenyl as sole carbon and energy sources by means of the hbp regulon . This regulon is composed of three genes, hbpCA and hbpD, coding for enzymes of a meta-cleavage pathway and the hbpR gene, which codes for a XylR/DmpR-type transcription regulator . It was previously shown that HbpR activates transcription from two sigma(54)-dependent promoters, P(hbpC) and P(hbpD), in the presence of 2-HBP . In this study, by using gel mobility shift assays with a purified fusion protein containing calmodulin binding protein (CBP) and HbpR, we detected two binding regions for HbpR in P(hbpC) and one binding region in P(hbpD) . DNase I footprints of the proximal binding region of P(hbpC) and of the binding region in P(hbpD) showed that CBP-HbpR protected a region composed of two inverted repeat sequences which were homologous to the binding sites identified for XylR . Unlike the situation in the XylR/P(u) system, we observed simultaneous binding of CBP-HbpR on the two upstream activating sequences (UASs) . Fragments with only one UAS did not show an interaction with HbpR, indicating that both pairs of UASs are needed for HbpR binding . The addition of both ATP and 2-HBP increased the DNA binding affinity of HbpR . These results showed for the first time that, for regulators of the XylR/DmpR type, the effector positively affects the recruitment of the regulatory protein on the enhancer DNA. Microbiol Res, 2002, 157(2), 93 - 102 Evidence for genotypic differences between the two siderovars of Pseudomonas tolaasii, cause of brown blotch disease of the cultivated mushroom Agaricus bisporus; Munsch P et al.; Sixteen representative isolates of Pseudomonas tolaasii, the causal agent of brown blotch of the cultivated mushroom Agaricus bisporus, were previously assigned to two siderovars (sv1 and sv2) on the basis of pyoverdines synthesized . Each isolate was pathogenic and produced a typical white line precipitate when cultured adjacent to Pseudomonas "reactans" strain LMG 5329 . These 16 isolates of P . tolaasii, representing sv1 and sv2, were further characterized using genotypic methods to examine the relationships between the isolates . Rep-PCR studies revealed two distinct patterns from these isolates, which were consistent with the siderovar grouping . Ribotyping differentiated P . tolaasii LMG 2342T (sv1) and PS 3a (sv2) into two distinct ribotypes . A pair of primers, targeted to a 2.1-kb fragment of tl1 (encoding a tolaasin peptide synthetase), yielded the same PCR product from P . tolaasii LMG 2342T (sv1) and PS 22.2 (sv1), but not from PS 3a (sv2) . Southern blot analysis indicated that homologues of tl1 are present in PS 3a, but the pattern of hybridization differed from PS 22.2 and LMG 2342T . Sequence determination and analysis of the internally transcribed spacer region ITSI for P . tolaasii LMG 2342T, LMG 6641, and PS 3a strains further supported the presence of the two siderovars . It is concluded that considerable genotypic differences exist among Finnish isolates of P . tolaasii causing brown blotch disease on the cultivated mushroom, which is in agreement with the phenotypic diversity highlighted through previous siderotyping studies. Pediatr Pulmonol, 2002 Jun, 33(6), 483 - 91 Predictors of deterioration of lung function in cystic fibrosis; Schaedel C et al.; The severity of lung disease in cystic fibrosis (CF) may be related to the type of mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and to environmental and immunological factors . Since pulmonary disease is the main determinant of morbidity and mortality in CF, it is important to identify factors that can explain and predict this variation . The aim of this longitudinal study of the whole Swedish CF population over age 7 years was to correlate genetic and clinical data with the rate of decline in pulmonary function . The statistical analysis was performed using the mixed model regression method, supplemented with calculation of relative risks for severe lung disease in age cohorts.The severity of pulmonary disease was to some extent predicted by CFTR genotype . Furthermore, the present investigation is the first long-term study showing a significantly more rapid deterioration of lung function in patients with concomitant diabetes mellitus . Besides diabetes mellitus, pancreatic insufficiency and chronic Pseudomonas colonization were found to be negative predictors of pulmonary function . In contrast to several other reports, we found no significant differences in lung function between genders . Patients with pancreatic sufficiency have no or only a slight decline of lung function with age once treatment is started, but an early diagnosis in this group is desirable . Plant J, 2002 May, 30(3), 361 - 71 The R1 gene for potato resistance to late blight (Phytophthora infestans) belongs to the leucine zipper/NBS/LRR class of plant resistance genes; Ballvora A et al.; Late blight caused by the oomycete Phytophthora infestans is the most destructive disease in potato cultivation worldwide . New, more virulent P . infestans strains have evolved which overcome the genetic resistance that has been introgressed by conventional breeding from wild potato species into commercial varieties . R genes (for single-gene resistance) and genes for quantitative resistance to late blight are present in the germplasm of wild and cultivated potato . The molecular basis of single-gene and quantitative resistance to late blight is unknown . We have cloned R1, the first gene for resistance to late blight, by combining positional cloning with a candidate gene approach . The R1 gene is member of a gene family . It encodes a protein of 1293 amino acids with a molecular mass of 149.4 kDa . The R1 gene belongs to the class of plant genes for pathogen resistance that have a leucine zipper motif, a putative nucleotide binding domain and a leucine-rich repeat domain . The most closely related plant resistance gene (36% identity) is the Prf gene for resistance to Pseudomonas syringae of tomato . R1 is located within a hot spot for pathogen resistance on potato chromosome V . In comparison to the susceptibility allele, the resistance allele at the R1 locus represents a large insertion of a functional R gene. Trends Plant Sci, 2002 May, 7(5), 210 - 6 Priming in plant-pathogen interactions; Conrath U et al.; Plants can acquire enhanced resistance to pathogens after treatment with necrotizing attackers, nonpathogenic root-colonizing pseudomonads, salicylic acid, beta-aminobutyric acid and many other natural or synthetic compounds . The induced resistance is often associated with an enhanced capacity to mobilize infection-induced cellular defence responses - a process called 'priming' . Although the phenomenon has been known for years, most progress in our understanding of priming has been made only recently . These studies show that priming often depends on the induced disease resistance key regulator NPR1 (also known as NIM1 or SAI1) and that priming has a major effect on the regulation of cellular plant defence responses. J Biol Chem, 2002 Jul 12, 277(28), 25096 - 105 Epub 2002 May 01. Isolation of cyanophycin-degrading bacteria, cloning and characterization of an extracellular cyanophycinase gene (cphE) from Pseudomonas anguilliseptica strain BI . The cphE gene from P . anguilliseptica BI encodes a cyanophycinhydrolyzing enzyme; Obst M et al.; Eleven bacteria capable of utilizing cyanophycin (cyanophycin granule polypeptide (CGP)) as a carbon source for growth were isolated . One isolate was taxonomically affiliated as Pseudomonas anguilliseptica strain BI, and the extracellular cyanophycinase (CphE) was studied because utilization of cyanophycin as a carbon source and extracellular cyanophycinases were hitherto not described . CphE was detected in supernatants of CGP cultures and purified from a corresponding culture of strain BI employing chromatography on the anion exchange matrix Q-Sepharose and on an arginine-agarose affinity matrix . The mature form of the inducible enzyme consisted of one type of subunit with M(r) = 43,000 and exhibited high specificity for CGP, whereas proteins and synthetic polyaspartic acid were not hydrolyzed or were only marginally hydrolyzed . Degradation products of the enzyme reaction were identified as aspartic acid-arginine dipeptides (beta-Asp-Arg) by high performance liquid chromatography and electrospray ionization mass spectrometry . The corresponding gene (cphE, 1254 base pairs) was identified in subclones of a cosmid gene library of strain BI by heterologous active expression in Escherichia coli, and its nucleotide sequence was determined . The enzyme exhibited only 27-28% amino acid sequence identity to intracellular cyanophycinases occurring in cyanobacteria . Analysis of the amino acid sequence of cphE revealed a putative catalytic triad consisting of the motif GXSXG plus a histidine and most probably a glutamate residue . In addition, the strong inhibition of the enzyme by Pefabloc((R)) and phenylmethylsulfonyl fluoride indicated that the catalytic mechanism of CphE is related to that of serine type proteases . Quantitative analysis on the release of beta-Asp-Arg dipeptides from C-terminal labeled CGP gave evidence for an exo-degradation mechanism. Sheng Wu Gong Cheng Xue Bao, 2002 Jan, 18(1), 45 - 50 {Nucleotide sequence and protein sequence analysis of GL-7-ACA acylase from Pseudomonas sp . 130}; Mao X et al.; The nucleotide sequence and N-, C-terminal amino acid sequences of alpha,beta-subunit of glutaryl 7-ACA acylase C130 from Pseudomonas sp . 130 were determined . The alignment of the acylase C130 with the other acylases shows that it has high homology with the acylases from Pseudomonas sp . GK16 and C427, but low homology with the others . There is large difference in the N-terminal of alpha-subunit, while the N-terminal of beta-subunit has significant conservation. Appl Environ Microbiol, 2002 May, 68(5), 2600 - 4 Role of glucose in enhancing the temperature-dependent growth inhibition of Escherichia coli O157:H7 ATCC 43895 by a Pseudomonas sp; Samelis J et al.; Growth of Escherichia coli O157:H7 strain ATCC 43895 was monitored at 5, 10, 15, and 25 degrees C in both pure and mixed (1:1) cultures with a gluconate-producing Pseudomonas sp . found in meat to evaluate the effect of the absence and presence of 1% glucose in broth on temperature-dependent competition . The number of colonies of the Pseudomonas strain exceeded 9 log CFU/ml under all conditions tested . The pathogen grew better as the temperature increased from 10 to 15 and 25 degrees C and grew better in pure culture than in mixed cultures . Pseudomonas sp . inhibited E . coli O157:H7 in cocultures with glucose at 10 degrees C, while at 15 degrees C the pathogen exhibited a biphasic pattern of growth with an intermediate inactivation period . Pathogen inhibition was much weaker in cocultures grown without glucose at 10 to 15 degrees C and, irrespective of glucose, at 25 degrees C . These results indicate that glucose enhances the growth inhibition of E . coli O157:H7 by some Pseudomonas spp., potentially due to its rapid uptake and conversion to gluconate, at low (< or = 15 degrees C) temperatures. Appl Environ Microbiol, 2002 May, 68(5), 2368 - 75 Transformation of isopropylamine to L-alaninol by Pseudomonas sp . strain KIE171 involves N-glutamylated intermediates; de Azevedo Wasch SI et al.; Pseudomonas sp . strain KIE171 was able to grow with isopropylamine or L-alaninol {S-(+)-2-amino-1-propanol} as the sole carbon source, but not with D-alaninol . To investigate the hypothesis that L-alaninol is an intermediate in the degradation of isopropylamine, two mini-Tn5 mutants unable to utilize both isopropylamine and L-alaninol were isolated . Whereas mutant KIE171-BI transformed isopropylamine to L-alaninol, mutant KIE171-BII failed to do so . The two genes containing a transposon insertion were cloned, and the DNA regions flanking the insertions were sequenced . Two clusters, one comprising eight ipu (isopropylamine utilization) genes (ipuABCDEFGH) and the other encompassing two genes (ipuI and orf259), were identified . Comparisons of sequences of the deduced Ipu proteins and those in the database suggested that isopropylamine is transported into the cytoplasm by a putative permease, IpuG . The next step, the formation of gamma-glutamyl-isopropylamide from isopropylamine, ATP, and L-glutamate, was shown to be catalyzed by IpuC, a gamma-glutamylamide synthetase . gamma-Glutamyl-isopropylamide is then subjected to stereospecific monooxygenation by the hypothetical four-component system IpuABDE, thereby yielding gamma-glutamyl-L-alaninol {gamma(L-glutamyl)-L-hydroxy-isopropylamide} . Enzymatic hydrolysis by a hydrolase, IpuF, was shown to finally liberate L-alaninol and to regenerate L-glutamate . No gene(s) encoding an enzyme for the next step in the degradation of isopropylamine was found in the ipu clusters . Presumably, L-alaninol is oxidized by an alcohol dehydrogenase to yield L-2-aminopropionaldehyde or it is deaminated by an ammonia lyase to propionaldehyde . Genetic evidence indicated that the aldehyde formed is then further oxidized by the hypothetical aldehyde dehydrogenases IpuI and IpuH to either L-alanine or propionic acid, compounds which can be processed by reactions of the intermediary metabolism. Appl Environ Microbiol, 2002 May, 68(5), 2179 - 87 Genes from Pseudomonas sp . strain BS involved in the conversion of L-2-amino-Delta(2)-thiazolin-4-carbonic acid to L-cysteine; Shiba T et al.; DL-2-amino-Delta(2)-thiazolin-4-carbonic acid (DL-ATC) is a substrate for cysteine synthesis in some bacteria, and this bioconversion has been utilized for cysteine production in industry . We cloned a DNA fragment containing the genes involved in the conversion of L-ATC to L-cysteine from Pseudomonas sp . strain BS . The introduction of this DNA fragment into Escherichia coli cells enabled them to convert L-ATC to cysteine via N-carbamyl-L-cysteine (L-NCC) as an intermediate . The smallest recombinant plasmid, designated pTK10, contained a 2.6-kb insert DNA fragment that has L-cysteine synthetic activity . The nucleotide sequence of the insert DNA revealed that two open reading frames (ORFs) encoding proteins with molecular masses of 19.5 and 44.7 kDa were involved in the L-cysteine synthesis from DL-ATC . These ORFs were designated atcB and atcC, respectively, and their gene products were identified by overproduction of proteins encoded in each ORF and by the maxicell method . The functions of these gene products were examined using extracts of E . coli cells carrying deletion derivatives of pTK10 . The results indicate that atcB and atcC are involved in the conversion of L-ATC to L-NCC and the conversion of L-NCC to cysteine, respectively . atcB was first identified as a gene encoding an enzyme that catalyzes thiazolin ring opening . AtcC is highly homologous with L-N-carbamoylases . Since both enzymes can only catalyze the L-specific conversion from L-ATC to L-NCC or L-NCC to L-cysteine, it is thought that atcB and atcC encode L-ATC hydrolase and N-carbamyl-L-cysteine amidohydrolase, respectively. J Biol Chem, 2002 Jun 28, 277(26), 23414 - 9 Epub 2002 Apr 24. Trifluoroethanol increases the stability of Delta(5)-3-ketosteroid isomerase . 15N NMR relaxation studies; Yun S et al.; In the equilibrium unfolding process of Delta(5)-3-ketosteroid isomerase from Pseudomonas testosteroni by urea, it was observed that the enzyme stability increases by 2.5 kcal/mol in the presence of 5% trifluoroethanol (TFE) . To elucidate the increased enzyme stability by TFE, the backbone dynamics of Delta(5)-3-ketosteroid isomerase were studied in the presence and absence of 5% TFE by (15)N NMR relaxation measurements, and the motional parameters (S(2), tau(e), and R(ex)) were extracted from the relaxation data using the model-free formalism . The presence of 5% TFE causes little change or a slight increase in the order parameters (S(2)) for a number of residues, which are located mainly in the dimer interface region . However, the majority of the residues exhibit reduced order parameters in the presence of 5% TFE, indicating that high frequency (pico- to nanosecond) motions are generally enhanced by TFE . The results suggest that the entropy can be an important factor for the enzyme stability, and the increase in entropy by TFE is partially responsible for the increased stability of Delta(5)-3-ketosteroid isomerase. Genetics, 2002 Apr, 160(4), 1661 - 71 Isolation and characterization of broad-spectrum disease-resistant Arabidopsis mutants; Maleck K et al.; To identify Arabidopsis mutants that constitutively express systemic acquired resistance (SAR), we constructed reporter lines expressing the firefly luciferase gene under the control of the SAR-inducible PR-1 promoter (PR-1/luc) . After EMS mutagenesis of a well-characterized transgenic line, we screened 250,000 M(2) plants for constitutive expression of the reporter gene in vivo . From a mutant collection containing several hundred putative mutants, we concentrated on 16 mutants lacking spontaneous hypersensitive response (HR) cell death . We mapped 4 of these constitutive immunity (cim) mutants to chromosome arms . Constitutive expression of disease resistance was established by analyzing responses to virulent Peronospora parasitica and Pseudomonas syringae strains, by RNA blot analysis for endogenous marker genes, and by determination of salicylic acid levels in the mutants . The variety of the cim phenotypes allowed us to define distinct steps in both the canonical SAR signaling pathway and a separate pathway for resistance to Erysiphe cichoracearum, active in only a subset of the mutants. Plant Cell, 2002 Apr, 14(4), 817 - 31 Tomato transcription factors pti4, pti5, and pti6 activate defense responses when expressed in Arabidopsis; Gu YQ et al.; The Pti4, Pti5, and Pti6 proteins from tomato were identified based on their interaction with the product of the Pto disease resistance gene, a Ser-Thr protein kinase . They belong to the ethylene-response factor (ERF) family of plant-unique transcription factors and bind specifically to the GCC-box cis element present in the promoters of many pathogenesis-related (PR) genes . Here, we show that these tomato ERFs are localized to the nucleus and function in vivo as transcription activators that regulate the expression of GCC box-containing PR genes . Expression of Pti4, Pti5, or Pti6 in Arabidopsis activated the expression of the salicylic acid-regulated genes PR1 and PR2 . Expression of jasmonic acid- and ethylene-regulated genes, such as PR3, PR4, PDF1.2, and Thi2.1, was affected differently by each of the three tomato ERFs, with Arabidopsis-Pti4 plants having very high levels of PDF1.2 transcripts . Exogenous application of salicylic acid to Arabidopsis-Pti4 plants suppressed the increased expression of PDF1.2 but further stimulated PR1 expression . Arabidopsis plants expressing Pti4 displayed increased resistance to the fungal pathogen Erysiphe orontii and increased tolerance to the bacterial pathogen Pseudomonas syringae pv tomato . These results indicate that Pti4, Pti5, and Pti6 activate the expression of a wide array of PR genes and play important and distinct roles in plant defense. Plant J, 2002 Apr, 30(1), 61 - 70 The Arabidopsis gain-of-function mutant dll1 spontaneously develops lesions mimicking cell death associated with disease; Pilloff RK et al.; We describe the characterization of a novel gain-of-function Arabidopsis mutant, dll1 (disease-like lesions1), which spontaneously develops lesions mimicking bacterial speck disease and constitutively expresses biochemical and molecular markers associated with pathogen infection . Despite the constitutive expression of defense-related responses, dll1 is unable to suppress the growth of virulent pathogens . However, dll1 elicits normal hypersensitive response in response to avirulent pathogens, thus indicating that dll1 is not defective in the induction of normal resistance responses . The lesion+ leaves of dll1 support the growth of hrcC mutant of Pseudomonas syringae, which is defective in the transfer of virulence factors into the plant cells, and therefore non-pathogenic to wild-type Col-0 plants . This suggests that dll1 intrinsically expresses many of the cellular processes that are required for pathogen growth during disease . Epistasis analyses reveal that salicylic acid and NPR1 are required for lesion formation, while ethylene modulates lesion development in dll1, suggesting that significant overlap exist between the signalling pathways leading to resistance- and disease-associated cell death . Our results suggest that host cell death during compatible interactions, at least in part, is genetically controlled by the plant and DLL1 may positively regulate this process. Mol Microbiol, 2002 Apr, 44(1), 73 - 88 Identification of Pseudomonas syringae pv . tomato genes induced during infection of Arabidopsis thaliana; Boch J et al.; Phytopathogenic bacteria possess a large number of genes that allow them to grow and cause disease on plants . Many of these genes should be induced when the bacteria come in contact with plant tissue . We used a modified in vivo expression technology (IVET) approach to identify genes from the plant pathogen Pseudomonas syringae pv . tomato that are induced upon infection of Arabidopsis thaliana and isolated over 500 in planta-expressed (ipx) promoter fusions . Sequence analysis of 79 fusions revealed several known and potential virulence genes, including hrp/hrc, avr and coronatine biosynthetic genes . In addition, we identified metabolic genes presumably important for adaptation to growth in plant tissue, as well as several genes with unknown function that may encode novel virulence factors . Many ipx fusions, including several corresponding to novel genes, are dependent on HrpL, an alternative RNA polymerase sigma factor that regulates the expression of virulence genes . Expression analysis indicated that several ipx fusions are strongly induced upon inoculation into plant tissue . Disruption of one ipx gene, conserved effector locus (CEL) orf1, encoding a putative lytic murein transglycosylase, resulted in decreased virulence of P . syringae . Our results demonstrate that this screen can be used successfully to isolate genes that are induced in planta, including many novel genes potentially involved in pathogenesis. Fiziol Zh, 2001, 47(6), 55 - 7 {The protective effect of anti-Pseudomonas blood preparations}; Nazarchuk LV et al.; Human blood antipseudomonas preparation activity has been studied on the model of Ps . Aeruginosa etiology sepsis in white not pedigree mice . It has been found out that antipseudomonas plasma and antipseudomonas immunoglobulin with antibody tire 1:80 possess marked therapeutic properties . The obtained results of the experimental studies are the grounds for clinical studies carrying out with the use of blood antipseudomonas preparations in combined therapy of the patients with diseases of Ps . Aeruginosa etiology. J Appl Physiol, 2002 May, 92(5), 2169 - 76 Characterization of LPS-induced lung inflammation in cftr-/- mice and the effect of docosahexaenoic acid; Freedman SD et al.; The mechanism by which Pseudomonas causes excessive inflammation in the cystic fibrosis lung is unclear . We have reported that arachidonic acid is increased and docosahexaenoic acid (DHA) decreased in lung, pancreas, and ileum from cftr-/- mice . Oral DHA corrected this defect and reversed the pathology . To determine which mediators regulate inflammation in lungs from cftr-/- mice and whether inhibition occurs with DHA, cftr-/- and wild-type (WT) mice were exposed to aerosolized Pseudomonas lipopolysaccharide (LPS) . After 2 days of LPS, tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2, and KC levels in bronchoalveolar lavage fluid were increased in cftr-/- compared with WT mice and not suppressed by pretreatment with oral DHA . Neutrophil levels were not different between cftr-/- and WT mice . After 3 days of aerosolized LPS, neutrophil concentration, TNF-alpha, and the eicosanoids 6-keto-PGF1alpha, PGF2alpha, PGE2, and thromboxane B2 were all increased in bronchoalveolar lavage fluid from cftr-/- mice compared with WT controls . Oral DHA had no significant effect on TNF-alpha levels in cftr-/- mice . In contrast, neutrophils and eicosanoids were decreased in cftr-/- but not in WT mice treated with DHA, indicating that the effects of DHA on these inflammatory parameters may be related to correction of the membrane lipid defect. J Mol Evol, 2002 Apr, 54(4), 437 - 57 A phylogenomic study of the OCTase genes in Pseudomonas syringae pathovars: the horizontal transfer of the argK-tox cluster and the evolutionary history of OCTase genes on their genomes; Sawada H et al.; Phytopathogenic Pseudomonas syringae is subdivided into about 50 pathovars due to their conspicuous differentiation with regard to pathogenicity . Based on the results of a phylogenetic analysis of four genes (gyrB, rpoD, hrpL, and hrpS), Sawada et al . (1999) showed that the ancestor of P . syringae had diverged into at least three monophyletic groups during its evolution . Physical maps of the genomes of representative strains of these three groups were constructed, which revealed that each strain had five rrn operons which existed on one circular genome . The fact that the structure and size of genomes vary greatly depending on the pathovar shows that P . syringae genomes are quite rich in plasticity and that they have undergone large-scale genomic rearrangements . Analyses of the codon usage and the GC content at the codon third position, in conjunction with phylogenomic analyses, showed that the gene cluster involved in phaseolotoxin synthesis (argK-tox cluster) expanded its distribution by conducting horizontal transfer onto the genomes of two P . syringae pathovars (pv . actinidiae and pv . phaseolicola) from bacterial species distantly related to P . syringae and that its acquisition was quite recent (i.e., after the ancestor of P . syringae diverged into the respective pathovars) . Furthermore, the results of a detailed analysis of argK {an anabolic ornithine carbamoyltransferase (anabolic OCTase) gene}, which is present within the argK-tox cluster, revealed the plausible process of generation of an unusual composition of the OCTase genes on the genomes of these two phaseolotoxin-producing pathovars: a catabolic OCTase gene (equivalent to the orthologue of arcB of P . aeruginosa) and an anabolic OCTase gene (argF), which must have been formed by gene duplication, have first been present on the genome of the ancestor of P . syringae; the catabolic OCTase gene has been deleted; the ancestor has diverged into the respective pathovars; the foreign-originated argK-tox cluster has horizontally transferred onto the genomes of pv . actinidiae and pv . phaseolicola; and hence two copies of only the anabolic OCTase genes (argK and argF) came to exist on the genomes of these two pathovars . Thus, the horizontal gene transfer and the genomic rearrangement were proven to have played an important role in the pathogenic differentiation and diversification of P . syringae. Cell, 2002 Mar 22, 108(6), 743 - 54 RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis; Mackey D et al.; In Arabidopsis, RPM1 confers resistance against Pseudomonas syringae expressing either of two sequence unrelated type III effectors, AvrRpm1 or AvrB . An RPM1-interacting protein (RIN4) coimmunoprecipitates from plant cell extracts with AvrB, AvrRpm1, or RPM1 . Reduction of RIN4 protein levels inhibits both the hypersensitive response and the restriction of pathogen growth controlled by RPM1 . RIN4 reduction causes diminution of RPM1 . RIN4 reduction results in heightened resistance to virulent Peronospora parasitica and P . syringae, and ectopic defense gene expression . Thus, RIN4 positively regulates RPM1-mediated resistance yet is, formally, a negative regulator of basal defense responses . AvrRpm1 and AvrB induce RIN4 phosphorylation . This may enhance RIN4 activity as a negative regulator of plant defense, facilitating pathogen growth . RPM1 may "guard" against pathogens that use AvrRpm1 and AvrB to manipulate RIN4 activity. Mol Plant Microbe Interact, 2002 Mar, 15(3), 281 - 91 Functional analyses of the Pto resistance gene family in tomato and the identification of a minor resistance determinant in a susceptible haplotype; Chang JH et al.; Pto is a member of a multigene family and encodes a serine/threonine kinase that mediates gene-for-gene resistance to strains of Pseudomonas syringae pv . tomato expressing avrPto . The inferred amino acid sequence of the Pto homologs from both resistant (LpimPth2 to LpimPth4) and susceptible (LescFen, LescPth2 to LescPth5) haplotypes suggested that most could encode functional serine/threonine kinases . In addition, the activation segments of the homologs are similar in sequence to that of Pto, and some have residues previously identified as required for binding of AvrPto by Pto in the yeast two-hybrid system . The Pto homologs were therefore characterized for transcription, for the ability of their products to interact with AvrPto in the yeast two-hybrid system, for their autophosphorylation activity, and for their potential to elicit cell death in the presence of and absence of a ligand, as well as their dependence on Prf . LpimPth5, LpimPth4, and LescPth4 were not transcribed at levels detectable by reverse transcription-polymerase chain reaction . The interaction with AvrPto was unique to Pto in the yeast two-hybrid system . LescPth2 autophosphorylated in vitro as a fusion protein . LpimPth2, LpimPth3, LpimPth4, LescPth3, and LescPth4 did not autophosphorylate in vitro . Transient expression of wild-type Fen and wild-type LpimPth3, as well as LescFen, LescPth3, and LescPth5 with perturbations in their P+1 loop caused cell death in Nicotiana benthamiana . LpimPth3 and LescPth3 with amino acid substitutions in the P+1 loop also elicited cell death in tomato; this was dependent on the presence of wild-type Prf . Consequently, some homologs could potentially encode functional resistance proteins . LescPth5 induced cell death specifically in response to expression of AvrPto in tobacco in a Prf-dependent manner; this is consistent with a homolog from a 'susceptible' haplotype encoding a minor recognition determinant. Plant Physiol, 2002 Apr, 128(4), 1313 - 22 Identification of Arabidopsis ethylene-responsive element binding factors with distinct induction kinetics after pathogen infection; Onate-Sanchez L et al.; Ethylene-responsive element binding factors (ERF) proteins are plant-specific transcription factors, many of which have been linked to stress responses . We have identified four Arabidopsis ERF genes whose expression was specifically induced by avirulent and virulent strains of the bacterial pathogen Pseudomonas syringae pv tomato, with overlapping but distinct induction kinetics . However, a delay in ERF mRNA accumulation after infection with the virulent strain was observed when compared with the avirulent strain . The induction of ERF gene expression in most cases preceded the mRNA accumulation of a basic chitinase gene, a potential downstream target for one or more of these ERFs . The expression of the ERF genes was examined among different Arabidopsis tissues, in response to the signaling molecules ethylene, methyl jasmonate, and salicylic acid (SA), and in Arabidopsis mutants with decreased or enhanced susceptibility to pathogens, and significant differences were observed . For example, in seedlings, some of the ERF genes were not induced by SA in the wild-type but were SA responsive in the pad4-1 mutant, suggesting that PAD4-1, which acts upstream of SA accumulation, is also involved in repressing the SA-induced expression of specific ERF genes . The four ERF proteins were shown to contain transcriptional activation domains . These results suggest that transcriptional activation cascades involving ERF proteins may be important for plant defense to pathogen attack and that some ERF family members could be involved in the cross-talk between SA- and jasmonic acid-signaling pathways. Clin Cancer Res, 2002 Apr, 8(4), 995 - 1002 Improved cytotoxic activity toward cell lines and fresh leukemia cells of a mutant anti-CD22 immunotoxin obtained by antibody phage display; Salvatore G et al.; Recombinant immunotoxins are fusion proteins composed of the Fv domains of antibodies fused to bacterial or plant toxins that are being developed for the targeted therapy of cancer . RFB4 (Fv)-Pseudomonas exotoxin 38 (PE38) is an immunotoxin that targets CD22 expressed on B cells and B-cell malignancies . A disulfide-stabilized form of RFB4 (Fv)-PE38 is being evaluated in a Phase I clinical trial . The aim of the present study was to improve the activity of RFB4 (Fv)-PE38 to more effectively treat patients with leukemias and lymphomas . To increase the affinity of RFB4 (Fv), we used the techniques of phage display and hot spot mutagenesis . We identified mutational hot spot sequences in heavy chain complementary determining region 3 (V(H) CDR3) and randomized these in a phage display library . Mutant phages were panned on CD22-positive Daudi cells . A variety of mutant Fvs were obtained, and the corresponding immunotoxins were prepared . Several mutant immunotoxins with increased binding affinity and cytotoxic activity were obtained . The most active immunotoxin contained amino acid residues Thr-His-Trp (THW) in place of Ser-Ser-Tyr (SSY) at positions 100, 100A, and 100B of the Fv and had an affinity improved from 85 nM to 6 nM . The THW mutant had a 5- to 10-fold increase in activity on various CD22-positive cell lines and was up to 50 times more cytotoxic to cells from patients with chronic lymphocytic leukemia and hairy-cell leukemia. J Microbiol Methods, 2002 Jun, 50(1), 97 - 100 A filtration, incubation and staining reactor including a new protocol for FISH; Poschen L et al.; A newly developed device for performing fluorescence in situ hybridization (FISH) is described . An adapted procedure was compared with two typical FISH protocols . Tests were performed with Pseudomonas cells and the gene probe EUB338 . With the novel procedure, we obtained a better recovery of cells and less variability in results. J Am Chem Soc, 2002 Apr 17, 124(15), 3814 - 5 Decay of the transient Cu(B)-CO complex is accompanied by formation of the heme Fe-CO complex of cytochrome cbb(3)-CO at ambient temperature: evidence from time-resolved Fourier transform infrared spectroscopy; Stavrakis S et al.; Time-resolved step-scan Fourier infrared spectroscopy has been used to study the CO-bound cbb(3)-type cytochrome c oxidase from Pseudomonas stutzeri at room temperature . We observe a single band in the FTIR spectrum at 1956 cm(-1) (beta-form) . The time-resolved data indicate that upon photolysis, CO is transferred from heme b(3) (nu(CO) = 1956 cm(-1)) to CuB (nu(CO) = 2064 cm(-1)) . The decay of the 2065 cm(-1) peak (t(1/2) = 120 +/- 16 ms) and the development of the 1956 cm(-1) peak (t(1/2) = 144 +/- 8 ms ) suggest that formation of the Fe-CO complex is concurrent with the decay of the CuB-CO complex . The intensity ratio of the Fe-CO/CuB-CO (2.15) remains constant for all data points, and thus we conclude that no fraction of CO escapes the binuclear center at 293 K. Structure (Camb), 2002 Apr, 10(4), 547 - 56 The structural basis for catalysis and specificity of the Pseudomonas cellulosa alpha-glucuronidase, GlcA67A; Nurizzo D et al.; Alpha-glucuronidases, components of an ensemble of enzymes central to the recycling of photosynthetic biomass, remove the alpha-1,2 linked 4-O-methyl glucuronic acid from xylans . The structure of the alpha-glucuronidase, GlcA67A, from Pseudomonas cellulosa reveals three domains, the central of which is a (beta/alpha)(8) barrel housing the catalytic apparatus . Complexes of the enzyme with the individual reaction products, either xylobiose or glucuronic acid, and the ternary complex of both glucuronic acid and xylotriose reveal a "blind" pocket which selects for short decorated xylooligosaccharides substituted with the uronic acid at their nonreducing end, consistent with kinetic data . The catalytic center reveals a constellation of carboxylates; Glu292 is poised to provide protonic assistance to leaving group departure with Glu393 and Asp365 both appropriately positioned to provide base-catalyzed assistance for inverting nucleophilic attack by water. Mol Cell Biochem, 2002 Jan, 229(1-2), 25 - 34 The impact of insulin-like growth factor-1 on the pattern of cardiac elongation factor-2 variants in a model of overload; Jager D et al.; Because of its key role in proteosynthesis, the total content of elongation factor-2 (EF-2) and the distribution of six main EF-2 variants were investigated after Pseudomonas Exotoxin A catalyzed {37P}ADP-ribosylation using 1D-PAGE and isoelectric focusing (IEF) in a rat model of hemodynamic overload with variable degrees of cardiac hypertrophy: Chronic NO-synthase inhibition by L-NAME (N-omega-nitro-L-arginine-methyl-ester; 0.75 mg/ml drinking water) induced arterial hypertension without hypertrophy but myocardial apoptosis; additional treatment with IGF-1 (osmotic micropumps) did not modify hypertension but reduced apoptosis allowing moderate hypertrophy of the left ventricles . Total EF-2 did not significantly increase in rats with hemodynamic overload with or without IGF-1 supplementation . A positive correlation was found between an acidic EF-2 variant and apoptosis (p = 0.01) . Hypertrophy under additional IGF-1 was combined with a shift of the EF-2 variants to basic subtypes (p < 0.01) . This finding may be indicative of the trophic potency of IGF-1. J Mol Microbiol Biotechnol, 2002 May, 4(3), 191 - 6 Control of temperature-responsive synthesis of the phytotoxin coronatine in Pseudomonas syringae by the unconventional two-component system CorRPS; Smirnova AV et al.; The phytopathogen Pseudomonas syringae produces the phytotoxin coronatine (COR) as a major virulence factor . COR and its precursor, coronafacic acid, function as molecular mimics of the plant signaling molecule jasmonate . A 32.8-kb plasmid-borne gene cluster mediates COR biosynthesis, which is optimal at 18 degrees C and non-detectable at 28 degrees C, the optimal growth temperature for P . syringae . The thermoregulation is mediated at the transcriptional level by an unconventional two-component regulatory system consisting of a histidine protein kinase, CorS, and two transcriptional activators, CorR and CorP . Dissection of this regulatory triad revealed that CorR binds to its target sequences in a thermoresponsive manner and that its DNA-binding activity is controlled by CorS . A Preliminary model for thermo-sensing by CorS is proposed based on its membrane topology and the analysis of translational fusions of CorS to reporter enzymes at different temperatures . CorP lacks a typical helix-turn-helix motif but possibly functions as a modulator of CorR or CorS activity . The thermoregulation of COR biosynthetic genes is widespread among various COR-producing P . syringae strains . Post-translational processes also contribute to the thermo-responsiveness of COR production . Additionally, COR synthesis in P . syringae is influenced by nutrient availability, rpoN encoding the alternative sigma factor sigma54, and HrpV, a negative regulator of hrp gene expression, suggesting a complex regulatory network governing phytotoxin synthesis. Int J Syst Evol Microbiol, 2002 Mar, 52(Pt 2), 513 - 23 Pseudomonas lini sp . nov., a novel species from bulk and rhizospheric soils; Delorme S et al.; The taxonomic position of eight fluorescent Pseudomonas strains isolated from bulk and rhizospheric soils, and from water was examined . These eight strains clustered in one phenon together with Pseudomomas mandelii (CFBP 4844T), but could still be differentiated from this type strain by four phenotypic features . The eight stains exhibited internal DNA-DNA hybridization values ranging from 60 to 100%, with deltaTm below 5 degrees C (3.9 and 4.3 degrees C) for the lowest values (60 and 66%) . The percentages of hybridization with type or reference strains of other Pseudomonas species tested ranged from 12 to 60% (deltaTm = 5.5 degrees C), indicating that the eight isolates studied constituted a discrete DNA homology group . Comparison of the 16S rDNA sequence of the strain representing this group (CFBP 5737T) with the sequences of other strains belonging to the genus Pseudomonas revealed that strain CFBP 5737T was a member of this genus and that these bacteria did not cluster with any previously described species of the genus Pseudomonas . The eight isolates belonged to two siderovars different from those described so far . On the basis of the results of phenotypic, DNA-DNA and phylogenetic analyses, and of siderotyping, a new species, Pseudomonas lini sp . nov . (type strain CFBP 5737T) is proposed. Ir Med J, 2002 Jan, 95(1), 14 - 6 Malignant otitis externa--a high index of suspicion is still needed for diagnosis; Walshe P et al.; Malignant otitis externa is a destructive inflammatory process of the petrous temporal bone which if untreated leads to osteomyelitis of the skull base and can be fatal . It is more common in immunocompromised and elderly insulin-dependant diabetic patients and is caused by infection with Pseudomonas species . Despite a range of laboratory and radiological tests it still remains difficult to diagnose, particularly in the early stages when it can be treated medically . We describe three cases which presented to this department in the past twelve months . In all cases the diagnosis was made clinically and confirmed per-operatively . Interestingly all three cases were relatively young patients who did not have an immunocompromised status and were not diabetic. J Basic Microbiol, 2002, 42(1), 75 - 9 Regulation of pyrimidine synthesis in Pseudomonas mendocina; Santiago MF et al.; Regulation of the de novo pyrimidine biosynthetic enzymes in the bacterium Pseudomonas mendocina was examined when its cells were grown on succinate as a carbon source . When P . mendocina was grown in the presence of orotic acid or uracil, the de novo enzyme activities were depressed with dihydroorotase activity being significantly depressed after uracil addition . Following pyrimidine limitation of a uracil auxotroph of P . mendocina deficient for orotate phosphoribosyltransferase activity, the pyrimidine biosynthetic pathway enzyme activities were affected indicating possible regulation at the level of enzyme synthesis . Of the de novo pathway enzymes assayed, dihydroorotate dehydrogenase exhibited the highest increase in its activity . The regulation of the pyrimidine biosynthetic pathway by pyrimidines in P . mendocina appeared similar to what was previously observed for the taxonomically-related species P . stutzeri. Protein Expr Purif, 2002 Apr, 24(3), 439 - 44 Tabtoxin-resistant protein: overexpression, purification, and characterization; Liu J et al.; One of the self-protection mechanisms in Pseudomonas syringae pv . tabaci, a pathogen of tobacco wildfire, is thought to be due to its tabtoxin-resistance gene (ttr) . In this study, the ttr gene was inserted into an expression vector, pQE30, and successfully expressed in Escherichia coli M15 at high levels . The purified recombinant tabtoxin-resistant protein (TTR) had an apparent molecular mass of about 21 kDa on SDS-PAGE as well as by mass spectroscopy and had a pI of 6.6 on isoelectric focusing-PAGE . Spectral analysis showed that TTR possesses a maximum fluorescence wavelength (lambda(max)) of 325 nm upon excitation at 282 nm and a positive band with a maximum at 195 nm and a broad negative band with a minimum at 215 nm in the far-UV CD spectrum . The spectrophotometric assay demonstrated the strong detoxification activity of TTR . These results are the first report of the characterization of the purified tabtoxin-resistant protein . Its capacity to detoxify tabtoxinine-beta-lactam shows that it must be one of the self-protection mechanisms in pv . tabaci . Mol Genet Genomics, 2002 Mar, 267(1), 16 - 26 Epub 2002 Feb 12. The tobacco bZIP transcription factor BZI-1 binds to G-box elements in the promoters of phenylpropanoid pathway genes in vitro, but it is not involved in their regulation in vivo; Heinekamp T et al.; Screening of a tobacco (Nicotiana tabacum) cDNA library resulted in the isolation of a clone encoding the bZIP transcription factor BZI-1 . With respect to amino acid sequence, conservation of protein domains, genomic exon-intron structure and expression pattern, BZI-1 is closely related to CPRF2, OHP1/2, BLZ1 and REB, a group of bZIP proteins which have been described in a number of dicot and monocot species . BZI-1 exhibits the characteristics of a transcription factor . It binds to G-box and C-box cis-elements in vitro, it is localised in the nucleus, and the N-terminal region of BZI-1 functions as an activation domain in both yeast and plant cells . Since BZI-1-related transcription factors have been isolated from dicots by in vitro binding to G-box elements in the chalcone synthase ( CHS) promoter, it has been suggested that phenylpropanoid pathway genes, such as CHS and PAL (phenylalanine ammonia-lyase), are targets of these proteins in vivo . However, after infection with Pseudomonas syringae or Tobacco Mosaic Virus, no changes in pathogen-induced PAL expression were observed in transgenic plants expressing increased levels of BZI-1 or a dominant negative form of the protein, BZI-1-DeltaN . In contrast to the tissue-specific expression of CHS and PAL, BZI-1 was found to be ubiquitously expressed in tobacco plants . Furthermore, no changes in the tissue-specific expression of PAL or CHS were observed in plants that were transgenic for BZI-1-DeltaN . Expression of a VP16-BZI-1 fusion protein would be expected to result in constitutive activation of the BZI-1 target genes . However, tetracycline-dependent expression of a VP16-BZI-1 protein in tobacco plants did not result in activation of CHS or PAL . On the basis of these data, we conclude that the phenylpropanoid pathway genes analysed are not targets of BZI-1 in vivo . Thus, the pattern of in vitro DNA binding of transcription factors need not always reflect their in vivo function. DNA Seq, 2001 Nov, 12(4), 281 - 4 Homology study of two polyhydroxyalkanoate (PHA) synthases from Pseudomonas aureofaciens; Umeda F et al.; Recently, we have cloned and analyzed two polyhydroxyalkanoate (PHA) synthase genes (phaC1 and phaC2 in the pha cluster) from Pseudomonas aureofaciens . In this report, the deduced amino acid (AA) sequences of PHA synthase 1 and PHA synthase 2 from P . aureofaciens are compared with those from three other bacterial strains (Pseudomonas sp . 61-3, P . oleovorans and P . aeruginosa) containing the homologous pha cluster . The level of homology of either PHA synthase 1 or PHA synthase 2 was high with each enzyme from these three bacterial strains . Furthermore, multialignment of PHA synthase AA sequences implied that both enzymes of PHA synthase 1 and PHA synthase 2 were highly conserved in the four strains including P . aureofaciens. J Comp Physiol {B}, 2002 Feb, 172(2), 163 - 8 The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate; Duman JG; Antifreeze proteins depress the freezing point of water while not affecting the melting point, producing a characteristic difference in freezing and melting points termed thermal hysteresis . Larvae of the beetle Dendroides canadensis accumulate potent antifreeze proteins (DAFPs) in their hemolymph and gut, but to achieve high levels of thermal hysteresis requires enhancers, such as glycerol . DAFPs have previously been shown to inhibit the activity of bacterial and hemolymph protein ice nucleators, however, the effect was not large and therefore the effectiveness of the DAFPs in promoting supercooling of the larvae in winter was doubtful . However, this study demonstrates that DAFPs, in combination with the thermal hysteresis enhancers glycerol (1 M) or citrate (0.5 M), eliminated the activity of hemolymph protein ice nucleators and Pseudomonas syringae ice-nucleating active bacteria, and lowered the supercooling points (nucleation temperatures) of aqueous solutions containing these ice nucleators to those of water or buffer alone . This shows that the DAFPs, along with glycerol, play a critical role in promoting hemolymph supercooling in overwintering D . canadensis . Also, DAFPs in combination with enhancers may be useful in applications which require inhibition of ice nucleators. Acta Crystallogr D Biol Crystallogr, 2002 Apr, 58(Pt 4), 697 - 9 Epub 2002 Mar 22. Crystallization and preliminary X-ray diffraction analysis of two pH-dependent forms of a di-haem cytochrome c peroxidase from Pseudomonas nautica; Dias JM et al.; Two crystal forms of cytochrome c peroxidase from Pseudomonas nautica were obtained, one at pH 4.0 using sodium citrate as precipitant and another at pH 5.3 using ammonium phosphate and sodium citrate as precipitants . The two forms belong to different space groups P3(1)21 (pH 4.0) and P6(4)22 (pH 5.3), with unit-cell parameters a = b = 114.5, c = 90.7 A and a = b = 151.0, c = 155.9 A, respectively . Several complete data sets were collected using synchrotron radiation at ESRF and Cu K(alpha) X-ray radiation from a rotating-anode generator . These results will contribute to clarifying the haem transitions occurring during peroxidatic reaction and the required electron-transfer processes and to elucidating the catalytic mechanism of the enzyme and the role of calcium in the activation process. J Bacteriol, 2002 Apr, 184(8), 2281 - 6 Global regulation by gidA in Pseudomonas syringae; Kinscherf TG et al.; Analysis of two virulence mutants of Pseudomonas syringae B728a revealed that the Tn 5 sites of insertion were within the gidA open reading frame (ORF) . These mutations were pleiotropic, affecting diverse phenotypic traits, such as lipodepsipeptide (syringomycin and syringopeptin) antibiotic production, swarming, presence of fluorescent pigment, and virulence . Site-specific recombination of a disrupted gidA gene into the chromosome resulted in the same phenotypic pattern as transposon insertion . Mutant phenotypes were restored by the gidA ORF on a plasmid . The salA gene, a copy number suppressor of the syringomycin-deficient phenotype in gacS and gacA mutants, was also found to suppress the antibiotic-negative phenotypes of gidA mutants, suggesting that gidA might play some role in salA regulation . Reporter studies with chromosomal salA-lacZ translational fusions confirmed that salA reporter expression decreased approximately fivefold in a gidA mutant background, with a concurrent decrease in the expression of the syringomycin biosynthetic reporter fusion syrB-lacZ . Wild-type levels of reporter expression were restored by supplying an intact gidA gene on a plasmid . Often described as being involved in cell division, more recent evidence suggests a role for gidA in moderating translational fidelity, suggesting a mechanism by which global regulation might occur . The gidA gene is essentially universal in the domains Bacteria and Eucarya but has no counterparts in Archaea, probably reflecting specific differences in the translational machinery between the former and latter domains. Microbiol Res, 2002, 157(1), 7 - 11 The white-line-in-agar test is not specific for the two cultivated mushroom associated pseudomonads, Pseudomonas tolaasii and Pseudomonas "reactans"; Munsch P et al.; A sharply defined white line in vitro forms between the pathogenic form of Pseudomonas tolaasii and another Pseudomonas bacterium, referred to as "reactans" . This interaction has been considered as highly specific . However, results presented in this study rise doubt about the strict specificity of this interaction, as some other pseudomonads, associated with the cultivated mushroom Agaricus bisporus, also yielded a white line precipitate when they were streaked towards Pseudomonas tolaasii LMG 2342T . Moreover, some Finnish isolates inducing brown blotch symptoms on mushrooms like P . tolaasii(T), produced a typical white precipitate when streaked towards P . "reactans" LMG5329, even though phenotypical and genotypical features exclude these isolates from the species P . tolaasii . We propose that the white-line-in-agar (WLA) test should no longer be considered as an unequivocal diagnostic trait of P . tolaasii. Biochim Biophys Acta, 2002 Feb 11, 1594(2), 207 - 18 The wild type bacterial Co(2+)/Co(2+)-phosphotriesterase shows a middle-range thermostability; Rochu D et al.; The phosphotriesterase (PTE) from Pseudomonas diminuta, a metalloenzyme that catalyses the hydrolysis of organophosphorus pesticides and nerve agents, has been described as a remarkably heat-stable protein {Grimsley et al., Biochemistry 36 (1997), 14366-14374} . Because substitution of the naturally occurring zinc ions by cobalt ions was found to enhance the enzyme catalytic activity, we investigated the thermal stability of the Co(2+)/Co(2+)-PTE . This study, carried out using capillary electrophoresis under optimised conditions in the pH range 9-10 compatible with optimal enzyme activity, provided evidence for irreversible denaturation according to the Lumry-Eyring model . A temperature-induced conformational transition (T(m) approximately equal to 58 degrees C) and an early growing of aggregates were observed . Comparison of UV spectra with heat-induced inactivation data clearly demonstrated that the PTE state populated above T(m) was neither native nor active . Differential scanning calorimetry showed only an exothermic trace due to aggregation of the denatured protein at T=76 degrees C . Accordingly, the temperature-induced denaturation process of the PTE could be described by a consecutive reaction model, including formation of an intermediate with enhanced activity at T approximately equal to 45 degrees C and an inactive unfolded state populated at T approximately equal to 58 degrees C, which leads to denatured aggregates . Thus, the wild type Co(2+)/Co(2+)-PTE displays a middle-range thermostability . Hence, for decontamination purposes under extreme Earth temperatures, wild type and engineered mutants of PTE substituted with other metal cations should be evaluated. Res Microbiol, 2002 Mar, 153(2), 89 - 98 Occurrence and properties of glutathione S-transferases in phenol-degrading Pseudomonas strains; Santos PM et al.; Pseudomonas sp . strains, able to degrade aromatic compounds such as phenol, were chosen to investigate the occurrence and characteristics of glutathione S-transferases (GSTs) . Affinity chromatography purification showed the presence of at least one GST in each studied strain . The purified proteins exhibited a great variety in the N-terminal sequences and different enzyme activities with the standard GST substrates tested . Two Pseudomonas strains, M1 and CF600, were chosen to investigate the GST activities under different growth conditions . Therefore, cells were grown either on phenol or on different nonaromatic carbon sources in the presence and absence of increasing phenol concentrations . In strain M1 a strong correlation between the activities of the catechol 1,2-dioxygenase and GST was observed in all the tested conditions . Moreover, growth on different organic acids also affected GST activity levels, with a negative correlation with the specific growth rate determined by each substrate . These results suggest a possible function of GST as a response to specific metabolic conditions determined by phenol toxicity and/or catabolism and the metabolic status of the cells . The same experiments performed with the CF600 strain did not show induction of GST activity in any of the tested conditions, indicating that GST_CF600 probably has a different role in cell metabolism . Native gel electrophoresis gave indications that GST dimerization could be an important process in the modulation of GST activity. Folia Microbiol (Praha), 2001, 46(5), 371 - 5 Multilocus enzyme electrophoresis analysis of Pseudomonas syringae pv . phaseolicola; Guven K; A multilocus enzyme electrophoresis technique was developed to detect variation in seven enzyme loci among isolates of Pseudomonas syringae pv . phaseolicola, representing three races from different geographical locations, the causal agent of the halo blight disease of beans . Cellulose acetate gel electrophoresis of seven enzymes revealed 19 electrotypes (ET) among 21 Pseudomonas syringae pv . phaseolicola isolates . One of the pathovar syringae and one of the pathovar tomato isolates were represented by two different ET . The population of Turkish isolates and three races of the pathovar phaseolicola appeared to be genetically diverse. Folia Microbiol (Praha), 2001, 46(6), 515 - 8 Production, purification and characterization of intracellular alanylaminopeptidase of Pseudomonas sp; Jankiewicz U et al.; The soil bacterium Pseudomonas sp . was found to synthesize an aminopeptidase that prefers Ala-beta-naphtylamide as substrate . The enzyme was purified 660-fold by ammonium sulfate fractionation, preparative electrophoresis, ion exchange chromatography on Protein-Pak Q 8 HR and molecular sieving chromatography on Zorbax SE-250 . When purified to homogeneity, the enzyme was shown to be a monomeric protein with a molar mass of 65 kDa; it showed a maximum activity at pH 7.5 and 45 degrees C. J Inorg Biochem, 2002 Feb, 88(3-4), 316 - 27 Sequential unfolding of the two-domain protein Pseudomonas stutzeri cytochrome c(4); Andersen NH et al.; P . stutzeri cytochrome c(4) is a di-haem protein, composed of two globular domains each with His-Met coordinated haem, and a hydrogen bond network between the domains . The domain foldings are highly symmetric but with specific differences including structural differences of ligand coordination, and different spin states of the oxidised haem groups . We have studied unfolding of oxidised P . stutzeri cyt c(4) induced thermally and by chemical denaturants . Horse heart cyt c was a reference molecule . Isothermal unfolding induced by guanidinium chloride and acid was followed by Soret, alpha/beta, and 701-nm band absorption, and by far-UV circular dichroism spectroscopy . Multifarious patterns emerge, but the two domains clearly unfold sequentially . One phase, assigned to unfolding of the N-terminal domain, proceeds at guanidinium concentrations up to approximately 1.0 M . This is followed by two overlapping phases at higher concentrations . The intermediate state maintains Fe-Met coordination, assigned to the C-terminal domain . Interdomain interaction is reflected in decreasing values of the cooperativity parameters . Differential scanning calorimetry shows a single peak, but two peaks appear when guanidinium chloride up to 0.4 M is present . This reflects different chemical action in chemical and thermal unfolding . Acid-induced unfolding kinetics was addressed by pH jumps using diode array stopped-flow techniques . Three kinetic phases in the 701 nm Fe-Met marker band, and four phases in the Soret and alpha/beta bands, spanning 4-1000 ms could be distinguished on pH jumps from 7.5 to the range 2.5-3.5 . In this range of time and pH cyt c appears to unfold in no more than two phases . Spectral properties of the kinetic intermediates could be identified . Sequential domain unfolding, formation of high-spin states, and an intermediate state with Fe-Met coordination to a single haem group are features of the unfolding kinetics. Bone Marrow Transplant, 2002 Feb, 29(4), 353 - 6 Radiologically guided fine needle lung biopsies in the evaluation of focal pulmonary lesions in allogeneic stem cell transplant recipients; Jantunen E et al.; Lung problems are common in allogeneic stem cell transplant (SCT) recipients . To evaluate the feasibility and diagnostic yield of radiologically guided fine needle lung biopsy (FNLB) in allogeneic SCT recipients with focal pulmonary lesions, a retrospective analysis was carried out . Between 1989 and 1998, radiologists performed a total of 30 FNLBs in 21 allogeneic SCT recipients, guided either by ultrasound (n = 17) or computed tomography (n = 13) . The median time from SCT to the first FNLB was 131 days (20-343 days) . Prophylactic platelet transfusions were given in 19 procedures (66%) . The complications of FNLB included clinically insignificant pneumothorax in four procedures (13%) and self-limiting haemoptysis in one case (3%) . The first FNLB was suggestive of invasive pulmonary aspergillosis (IPA) in five patients (24%) . Additional clinically useful findings of FNLB included Pseudomonas (two patients) and Nocardia (one patient) . The final diagnosis of pulmonary lesions was IPA in 14 patients, immunological lung problems in four patients and other in three patients . Radiologically guided FNLB is feasible in allogeneic SCT recipients and has a low complication rate . The diagnostic yield is high especially for IPA. Plant Physiol, 2002 Mar, 128(3), 1046 - 56 Benzothiadiazole-induced priming for potentiated responses to pathogen infection, wounding, and infiltration of water into leaves requires the NPR1/NIM1 gene in Arabidopsis; Kohler A et al.; Systemic acquired resistance (SAR) is a plant defense state that is induced, for example, after previous pathogen infection or by chemicals that mimic natural signaling compounds . SAR is associated with the ability to induce cellular defense responses more rapidly and to a greater degree than in noninduced plants, a process called "priming." Arabidopsis plants were treated with the synthetic SAR inducer benzothiadiazole (BTH) before stimulating two prominent cellular defense responses, namely Phe AMMONIA-LYASE (PAL) gene activation and callose deposition . Although BTH itself was essentially inactive at the immediate induction of these two responses, the pretreatment with BTH greatly augmented the subsequent PAL gene expression induced by Pseudomonas syringae pv . tomato infection, wounding, or infiltrating the leaves with water . The BTH pretreatment also enhanced the production of callose, which was induced by wounding or infiltrating the leaves with water . It is interesting that the potentiation by BTH pretreatment of PAL gene activation and callose deposition was not seen in the Arabidopsis nonexpresser of PR genes 1/noninducible immunity 1 mutant, which is compromised in SAR . In a converse manner, augmented PAL gene activation and enhanced callose biosynthesis were found, without BTH pretreatment, in the Arabidopsis constitutive expresser of pathogenesis-related genes (cpr)1 and constitutive expresser of pathogenesis-related genes 5 mutants, in which SAR is constitutive . Moreover, priming for potentiated defense gene activation was also found in pathogen-induced SAR . In sum, the results suggest that priming is an important cellular mechanism in acquired disease resistance of plants that requires the nonexpresser of PR genes 1/noninducible immunity 1 gene. Int J Med Microbiol, 2002 Feb, 291(6-7), 523 - 9 The ARTT motif and a unified structural understanding of substrate recognition in ADP-ribosylating bacterial toxins and eukaryotic ADP-ribosyltransferases; Han S et al.; ADP-ribosylation is a widely occurring and biologically critical covalent chemical modification process in pathogenic mechanisms, intracellular signaling systems, DNA repair, and cell division . The reaction is catalyzed by ADP-ribosyltransferases, which transfer the ADP-ribose moiety of NAD to a target protein with nicotinamide release . A family of bacterial toxins and eukaryotic enzymes has been termed the mono-ADP-ribosyltransferases, in distinction to the poly-ADP-ribosyltransferases, which catalyze the addition of multiple ADP-ribose groups to the carboxyl terminus of eukaryotic nucleoproteins . Despite the limited primary sequence homology among the different ADP-ribosyltransferases, a central cleft bearing the NAD-binding pocket formed by the two perpendicular beta-sheet cores has been remarkably conserved between bacterial toxins and eukaryotic mono- and poly-ADP-ribosyltransferases . The majority of bacterial toxins and eukaryotic mono-ADP-ribosyltransferases are characterized by conserved His and catalytic Glu residues . In contrast, diphtheria toxin, Pseudomonas exotoxin A, and eukaryotic poly-ADP-ribosytransferases are characterized by conserved Arg and catalytic Glu residues . Structural and mutagenic studies of the NAD-binding core of a binary toxin and a C3-like toxin identified an ARTT motif (ADP-ribosylating turn-turn motif) that is implicated in substrate specificity and recognition . Here we apply structure-based sequence alignment and comparative structural analyses of all known structures of ADP-ribosyltransfeases to suggest that this ARTT motif is functionally important in many ADP-ribosylating enzymes that bear a NAD-binding cleft as characterized by conserved Arg and catalytic Glu residues . Overall, structure-based sequence analysis reveals common core structures and conserved active sites of ADP-ribosyltransferases to support similar NAD-binding mechanisms but differing mechanisms of target protein binding via sequence variations within the ARTT motif structural framework . Thus, we propose here that the ARTT motif represents an experimentally testable general recognition motif region for many ADP-ribosyltransferases and thereby potentially provides a unified structural understanding of substrate recognition in ADP-ribosylation processes. J Bacteriol, 2002 Apr, 184(7), 1916 - 24 Two distinct alcohol dehydrogenases participate in butane metabolism by Pseudomonas butanovora; Vangnai AS et al.; The involvement of two primary alcohol dehydrogenases, BDH and BOH, in butane utilization in Pseudomonas butanovora (ATCC 43655) was demonstrated . The genes coding for BOH and BDH were isolated and characterized . The deduced amino acid sequence of BOH suggests a 67-kDa alcohol dehydrogenase containing pyrroloquinoline quinone (PQQ) as cofactor and in the periplasm (29-residue leader sequence) . The deduced amino acid sequence of BDH is consistent with a 70.9-kDa, soluble, periplasmic (37-residue leader sequence) alcohol dehydrogenase containing PQQ and heme c as cofactors . BOH and BDH mRNAs were induced whenever the cell's 1-butanol oxidation activity was induced . When induced with butane, the gene for BOH was expressed earlier than the gene for BDH . Insertional disruption of bdh or boh affected adversely, but did not eliminate, butane utilization by P . butanovora . The P . butanovora mutant with both genes boh and bdh inactivated was unable to grow on butane or 1-butanol . These cells, when grown in citrate and incubated in butane, developed butane oxidation capability and accumulated 1-butanol . The enzyme activity of BOH was characterized in cell extracts of the P . butanovora strain with bdh disrupted . Unlike BDH, BOH oxidized 2-butanol . The results support the involvement of two distinct NAD(+)-independent, PQQ-containing alcohol dehydrogenases, BOH (a quinoprotein) and BDH (a quinohemoprotein), in the butane oxidation pathway of P . butanovora. Can J Microbiol, 2002 Jan, 48(1), 49 - 59 Characterization of tdt genes for the degradation of tricyclic diterpenes by Pseudomonas diterpeniphila A19-6a; Morgan CA et al.; Resin acids are tricyclic diterpenes that are toxic to aquatic life when released in high concentrations in pulp mill effluents . These naturally formed organic acids are readily degraded by bacteria and fungi; nevertheless, many of the mechanisms involved are still unknown . We report the localization, cloning, and sequencing of genes for abietane degradation (9.18 kb; designated tdt (tricyclic diterpene) LRSABCD) from the gamma-Proteobacterium Pseudomonas diterpeniphila A19-6a . Using gene knockout mutants, we demonstrate that tdtL, encoding a putative CoA ligase, is required for growth on abietic and dehydroabietic acids . A second gene knockout in tdtD, encoding a putative cytochrome P450 monooxygenase, reduced the growth of strain A19-6a on abietic and dehydroabietic acids as sole sources of carbon and energy, but did not eliminate growth . The degree of homology between P450TdtD and P450TerpC, the closest known P450 homologue to TdtD, identifies TdtD as a new member of the P450 superfamily . Hybridization of six of the tdt genes to genomic DNA of a related resin acid degrading bacterium Pseudomonas abietaniphila BKME-9 identified tdt homologues in this strain that utilizes aromatic ring dioxygenase genes (dit) to open the ring structure of abietic and dehydroabietic acids . These results suggest the tdt and dit genes may function in concert to allow these Pseudomonas strains to degrade resin acids . Homologues of several of the tdt genes were detected in resin acid degrading Ralstonia and Comamonas species within the beta- and gamma-Proteobacteria. FEMS Microbiol Lett, 2002 Jan 22, 207(1), 75 - 80 Natural transformation of Pseudomonas stutzeri by single-stranded DNA requires type IV pili, competence state and comA; Meier P et al.; Pseudomonas stutzeri, in addition to being transformed by duplex DNA, is also transformed by the sense or antisense strand of the genetic marker employed (hisX(+)) or by heat-denatured chromosomal DNA . Transformation was absent in non-competent cells and in mutants defective for pilus biogenesis (pilA, pilC) and function (pilT) or DNA translocation into the cytoplasm (comA) . Uptake of (3)H-thymidine-labeled single-stranded DNA was hardly detectable reflecting the 20- to 60-fold lower transformation compared to duplex DNA . The results suggest that the steps in natural transformation also accommodate single-stranded DNA and that DNA translocation from the periplasm into the cytoplasm is not necessarily coupled to the degradation of a complementary strand . Small DNA single-stranded fragments are thus not excluded from horizontal gene transfer by transformation. Plant Cell, 2002 Feb, 14(2), 435 - 50 Large-scale structure-function analysis of the Arabidopsis RPM1 disease resistance protein; Tornero P et al.; The Arabidopsis RPM1 gene confers resistance against Pseudomonas syringae expressing either the AvrRpm1 or the AvrB type III effector protein . We present an exhaustive genetic screen for mutants that no longer recognize avrRpm1 . Using an inducible avrRpm1 expression system, we identified 110 independent mutations . These mutations represent six complementation groups . None discriminates between avrRpm1 and avrB recognition . We identified 95 rpm1 alleles and present a detailed structure--function analysis of the RPM1 protein . Several rpm1 mutants retain partial function, and we deduce that their residual activity is dependent on the level of avrRpm1 signal . In these mutants, the hypersensitive response remains activated if the signal goes above a certain threshold . Missense mutations in rpm1 are highly enriched in the nucleotide binding domain, suggesting that this region plays a key role either in the hypersensitive response associated with RPM1 activation or in RPM1 stability . Cluster analysis of rpm1 alleles defines functionally important residues that are highly conserved between nucleotide binding site leucine-rich repeat R proteins and those that are unique to RPM1 . Regions of RPM1 to which no loss-of-function alleles map may represent domains in which variation is tolerated and may contribute to the evolution of new R gene specificities. Curr Opin Mol Ther, 2002 Feb, 4(1), 72 - 5 Technology evaluation: BL22, NCI; Barth S; BL22 (RFB4(dsFv)-PE38) is a recombinant Pseudomonas exotoxin-based immunotoxin under development by the National Cancer Institute for the treatment of B-cell malignancies . It is composed of the disulfide-stabilized Fv portion of the anti-CD22 antibody RFB4 genetically fused to a truncated form of Pseudomonas exotoxin A . It has entered phase I trials for the treatment of B-cell lymphoma. OMICS, 2002, 6(1), 11 - 21 Physical mapping, BAC-end sequence analysis, and marker tagging of the soilborne nematicidal bacterium, Pseudomonas synxantha BG33R; Wechter WP et al.; A bacterial artificial chromosome (BAC) library was constructed for the genome of the rhizosphere-inhabiting fluorescent pseudomonad Pseudomonas synxantha BG33R . Three thousand BAC clones with an average insert size of 140 kbp and representing a 70-fold genomic coverage were generated and arrayed onto nylon membranes . EcoRI fingerprint analysis of 986 BAC clones generated 23 contigs and 75 singletons . Hybridization analysis allowed us to order the 23 contigs and condense them into a single contig, yielding an estimated genome size of 5.1 Mb for P . synxantha BG33R . A minimum-tile path of 47 BACs was generated and end-sequenced . The genetic loci involved in ring nematode egg-kill factor production in BG33R Tn5 mutants, 246 (vgrG homolog), 1122 (sensor kinase homolog), 1233 (UDP-galactose epimerase homolog), 1397 (ferrisiderophore receptor homolog), and 1917 (ribosomal subunit protein homolog), have been mapped onto the minimum-tile BAC library . Two of the genetic regions that flank Tn5 insertions in BG33R egg-kill-negative mutants 1233 and 1397 are separated by a single BAC clone . Fragments isolated by ligation-mediated PCR of the Tn5 mutagenized regions of 29 randomly selected, non-egg-kill-related, insertion mutants have been anchored onto the ordered physical map of P . synxantha. Appl Microbiol Biotechnol, 2002 Feb, 58(2), 229 - 36 Cloning, characterization and comparison of the Pseudomonas mendocina polyhydroxyalkanoate synthases Phac1 and PhaC2; Hein S et al.; This study describes a comparison of the polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 of Pseudomonas mendocina . The P mendocina pha gene locus, encoding two PHA synthase genes {phaC1Pm and phaC2pm flanking a PHA depolymerase gene (phaZ)}, was cloned, and the nucleotide sequences of phaC1Pm (1,677 bp), phaZ (1,034 bp), and phaC2pm (1,680 bp) were determined . The amino acid sequences deduced from phaC1Pm and phaC2pm showed highest similarities to the corresponding PHA synthases from other pseudomonads sensu stricto . The two PHA synthase genes conferred PHA synthesis to the PHA-negative mutants P . putida GPp104 and Ralstonia eutropha PHB-4 . In P . putida GPp 104, phaC1Pm and phaC2Pm mediated PHA synthesis of medium-chain-length hydroxyalkanoates (C6-C12) as often reported for other pseudomonads . In contrast, in R . eutropha PHB-4, either PHA synthase gene also led to the incorporation of 3-hydroxybutyrate (3HB) into PHA . Recombinant strains of R . eutropha PHB-4 harboring either P . mendocina phaC gene even accumulated a homopolyester of 3HB during cultivation with gluconate, with poly(3HB) amounting to more than 80% of the cell dry matter if phaC2 was expressed . Interestingly, recombinant cells harboring the phaC1 synthase gene accumulated higher amounts of PHA when cultivated with fatty acids as sole carbon source, whereas recombinant cells harboring PhaC2 synthase accumulated higher amounts when gluconate was used as carbon source in storage experiments in either host . Furthermore, isogenic phaC1 and phaC2 knock-out mutants of P . mendocina provided evidence that PhaC1 is the major enzyme for PHA synthesis in P . mendocina, whereas PhaC2 contributes to the accumulation of PHA in this bacterium to only a minor extent, and then only when cultivated on gluconate. J Vasc Surg, 2002 Mar, 35(3), 569 - 72 Melioidosis presenting as an infected intrathoracic subclavian artery pseudoaneurysm treated with femoral vein interposition graft; Schindler N et al.; We present the first case of in situ replacement of an infected subclavian artery using superficial femoral vein and the fourth reported case of an infected arterial pseudoaneurysm caused by pseudomonas pseudomallei . Sepsis and hoarseness developed in a 58-year-old man after recent travel to Borneo, Indonesia . Indirect laryngoscopy revealed a paralyzed right vocal cord . Computed tomography and arteriography revealed a 6.5-cm pseudoaneurysm of the proximal right subclavian artery . Blood cultures grew pseudomonas pseudomallei . An abnormal cardiac stress test prompted a coronary angiography, which revealed severe coronary artery disease.The patient underwent coronary artery bypass and in situ replacement of the infected subclavian artery pseudoaneurysm with a superficial femoral vein, along with placement of a pectoralis major muscle flap to cover the vein graft . Operative cultures of the pseudoaneurysm grew pseudomonas pseudomallei . The patient was treated with a 6-week course of intravenous ceftazidime and oral doxycycline and then continued on oral amoxicillin-clavulanate . One week after discontinuing intravenous antibiotics, the patient presented to the emergency department with a rapidly expanding, pulsatile mass in the right supraclavicular space . He was taken emergently to the operating room . After hypothermic circulatory arrest was accomplished, the disrupted vein graft and aneurysm cavity were resected and the subclavian artery was oversewn proximally and distally . Parenteral ceftazidime was continued for 3 months and oral amoxicillin-clavulanate (augmentin) was continued indefinitely . There was no evidence of infection clinically or by computed tomographic scan 2 years later . Although autogenous vein replacement of infected arteries and grafts may be successful in the majority of cases, this strategy should probably be avoided when particularly virulent bacteria such as the organism in this case are present. Appl Microbiol Biotechnol, 2002 Feb, 58(2), 138 - 46 The elusive roles of bacterial glutathione S-transferases: new lessons from genomes; Vuilleumier S et al.; Glutathione S-transferases constitute a large family of enzymes which catalyze the addition of glutathione to endogenous or xenobiotic, often toxic electrophilic chemicals . Eukaryotic glutathione S-transferases usually promote the inactivation, degradation or excretion of a wide range of compounds by formation of the corresponding glutathione conjugates . In bacteria, by contrast, the few glutathione S-transferases for which substrates are known, such as dichloromethane dehalogenase, 1,2-dichloroepoxyethane epoxidase and tetrachlorohydroquinone reductase, are catabolic enzymes with an essential role for growth on recalcitrant chemicals . Glutathione S-transferase genes have also been found in bacterial operons and gene clusters involved in the degradation of aromatic compounds . Information from bacterial genome sequencing projects now suggests that glutathione S-transferases are present in large numbers in proteobacteria . In particular, the genomes of three Pseudomonas species each include at least ten different glutathione S-transferase genes . Several of the corresponding proteins define new classes of the glutathione S-transferase family and may also have novel functions that remain to be elucidated. Khirurgiia (Mosk), 2002, (1), 34 - 5 {Immune status in diabetic patients with pyo-necrotic lesions of lower extremities}; Zemlianoi AB et al.; Long-term and severe pyonecrotic processes in diabetic patients testify to severe disorders of immune system in this disease . High titer of antibodies to tested autostrain demonstrated its etiologic role in infectious process . The study group consisted of 29 patients (with diabetic pyonecrotic foot lesions), control group--17 patients with burns of III a, b--IV stage affecting from 20 to 60% of body surface . In diabetic patients antibodies titer to the most encountered infectious agents Staphilococcus aureus and Pseudomonas aeroginosa was lower than in burn patients with immunity deficiency . Decrease of antibodies titer in diabetic patients testifies to high insufficiency of B-immunity. Hum Gene Ther, 2002 Mar 1, 13(4), 497 - 508 Gene therapy of murine solid tumors with T cells transduced with a retroviral vascular endothelial growth factor--immunotoxin target gene; Jin N et al.; Solid tumor growth can be inhibited by targeting its neovasculature with vascular endothelial growth factor (VEGF)-toxin fusion proteins (FPs), but these agents have been limited by their inability to localize at the tumor site . In this study, we devised a gene therapy approach intended to deliver VEGF-toxin directly to tumor . Antigen-specific cytotoxic T lymphocytes (CTLs) served as vehicles to deliver a retroviral VEGF-toxin fusion protein to its specific leukemia cell target in vivo . A retroviral vector was constructed for gene therapy with VEGF positioned downstream of its 27-amino acid leader sequence, which promoted secretion of a catalytic immunotoxin containing either truncated diphtheria toxin or Pseudomonas exotoxin A . VEGF was chosen on the basis of the expression of VEGF receptor on endothelial cells in the tumor neovasculature . The VEGF FP was first expressed and secreted by mammalian NIH 3T3 cells . Intracellular expression of both VEGF and toxin was verified by immunofluorescence . In vitro, supernatants collected from transfected cells specifically inhibited the growth of VEGF receptor-expressing human umbilical vein endothelial cells (HUVECs), but not a control cell line . In vivo findings correlated with in vitro findings . A retroviral vector containing the target gene and a nerve growth factor receptor (NGFR) reporter gene was used to transiently transduce T15, a CD8(+) CTL line that specifically recognizes C1498, a lethal C57BL/6 myeloid tumor . Transduced T15 cells injected intravenously significantly inhibited the growth of subcutaneous tumor, whereas nontransduced controls did not . Together, these data indicate that gene therapy of T cells with retrovirus containing a VEGF-immunotoxin target gene may be a valid means of inhibiting a broad range of solid tumors dependent on angiogenesis. J Mol Microbiol Biotechnol, 2002 Mar, 4(2), 151 - 61 Analysis of regulatory elements and genes required for carbon tetrachloride degradation in Pseudomonas stutzeri strain KC; Sepulveda-Torre L et al.; Previously, we described the generation and initial characterization of four Tn5 mutants of Pseudomonas stutzeri strain KC with impaired ability to degrade carbon tetrachloride (Sepulveda-Torres et al., 1999) . In this study, we show cloning and sequencing of an 8.3 kbp region in which all four transposons were located . This fragment encodes eight potential genes and is located in the central part of the 25 kbp fragment recently identified by Lewis et al . (2000) and shown by them to be sufficient to confer carbon tetrachloride transformation capability upon other pseudomonads . The four transposon insertion mutants mapped in ORF's F and I designated by Lewis et al . (2000) . This is consistent with the results by Lewis et al . (2000) that orfFis required for carbon tetrachloride degradation . We further established that orfl is required for CCl4 degradation since the three mutants in this ORF were unable to degrade carbon tetrachloride . We present our analysis of the gene and protein sequences from the 8.3 kbp region and propose a tentative model for the role of different genes in the synthesis and activity of pyridine-2,6-bis(thiocarboxylate) (PDTC), the secreted factor responsible for carbon tetrachloride dechlorination . We also found a putative promoter that overlaps with a Fur-box-like sequence in the region upstream of mutated genes . To test this putative promoter region and Fur-box, we generated and ligated DNA fragments containing wild-type and mutant Fur-boxes to a lacZ reporter . The wild-type fragment showed promoter activity that is regulated by the concentration of iron in the medium . Finally, we screened a selection of Pseudomonas strains, including P . putida DSMZ 3601--a strain known to produce PDTC--for the presence of the genes characterized in this study . None of the strains tested positive, suggesting that Pseudomonas stutzeri strain KC may possess a distinct biosynthetic pathway for PDTC production. Science, 2002 Mar 1, 295(5560), 1722 - 6 A functional screen for the type III (Hrp) secretome of the plant pathogen Pseudomonas syringae; Guttman DS et al.; Type III secreted "effector" proteins of bacterial pathogens play central roles in virulence, yet are notoriously difficult to identify . We used an in vivo genetic screen to identify 13 effectors secreted by the type III apparatus (called Hrp, for "hypersensitive response and pathogenicity") of the plant pathogen Pseudomonas syringae . Although sharing little overall homology, the amino-terminal regions of these effectors had strikingly similar amino acid compositions . This feature facilitated the bioinformatic prediction of 38 P . syringae effectors, including 15 previously unknown proteins . The secretion of two of these putative effectors was shown to be type III--dependent . Effectors showed high interstrain variation, supporting a role for some effectors in adaptation to different hosts. Appl Environ Microbiol, 2002 Mar, 68(3), 1403 - 7 Reduction of olive knot disease by a bacteriocin from Pseudomonas syringae pv . ciccaronei; Lavermicocca P et al.; A bacteriocin produced by Pseudomonas syringae pv . ciccaronei, used at different purification levels and concentrations in culture and in planta, inhibited the multiplication of P . syringae subsp . savastanoi, the causal agent of olive knot disease, and affected the epiphytic survival of the pathogen on the leaves and twigs of treated olive plants . Treatments with bacteriocin from P . syringae pv . ciccaronei inhibited the formation of overgrowths on olive plants caused by P . syringae subsp . savastanoi strains PVBa229 and PVBa304 inoculated on V-shaped slits and on leaf scars at concentrations of 10(5) and 10(8) CFU ml(-1), respectively . In particular, the application of 6,000 arbitrary units (AU) of crude bacteriocin (dialyzed ammonium sulfate precipitate of culture supernatant) ml(-1) at the inoculated V-shaped slits and leaf scars resulted in the formation of knots with weight values reduced by 81 and 51%, respectively, compared to the control, depending on the strains and inoculation method used . Crude bacteriocin (6,000 AU ml(-1)) was also effective in controlling the multiplication of epiphytic populations of the pathogen . In particular, the bacterial populations recovered after 30 days were at least 350 and 20 times lower than the control populations on twigs and on leaves, respectively . These results suggest that bacteriocin from P . syringae pv . ciccaronei can be used effectively to control the survival of the causal agent of olive knot disease and to prevent its multiplication at inoculation sites. J Appl Microbiol, 2002, 92(3), 517 - 25 Pyrimidine biosynthesis in Pseudomonas oleovorans; Haugaard LE et al.; AIMS: To investigate the regulation of de novo pyrimidine biosynthesis in the polyhydroxyalkanoate-producing bacterium Pseudomonas oleovorans at the level of enzyme synthesis and at the level of aspartate transcarbamoylase activity . METHODS AND RESULTS: The effect of pyrimidine supplementation on the pyrimidine biosynthetic pathway enzyme activities was analysed relative to carbon source . Two uracil auxotrophs of P . oleovorans were isolated that were deficient for aspartate transcarbamoylase or dihydroorotase activity . Pyrimidine limitation of these auxotrophs increased the de novo pathway activities to varying degrees depending on the pathway mutation and the carbon source utilized . At the level of aspartate transcarbamoylase activity, pyrophosphate and uridine ribonucleotides were found to be strongly inhibitory of the Ps . oleovorans enzyme . CONCLUSIONS: Pyrimidine biosynthesis is regulated in Ps . oleovorans . Taxonomically, the regulation of the pyrimidine biosynthetic pathway appeared dissimilar from previously studied Pseudomonas species . SIGNIFICANCE AND IMPACT OF THE STUDY: New insights regarding the regulation of nucleic acid metabolism are provided that could prove significant during the genetic manipulation of Ps . oleovorans to increase the synthesis of polyhydroxyalkanoates. Br J Cancer, 2002 Jan 21, 86(2), 285 - 91 IL-4 receptors on human medulloblastoma tumours serve as a sensitive target for a circular permuted IL-4-Pseudomonas exotoxin fusion protein; Joshi BH et al.; Cytotoxins directed to interleukin-4 receptors have shown to mediate relatively selective cytotoxicity against a variety of human cancer cells in vitro and in vivo . In an ongoing Phase I clinical trial, a recombinant protein comprised of circularly permuted IL-4 fused to a mutated form of Pseudomonas exotoxin (the fusion protein termed IL-4(38-37)-PE38KDEL or cpIL4-PE) has shown antitumour activity against malignant glioma . Human medulloblastomas are neuroectodermal tumours that occur in children and have a poor prognosis . The goal of this study was to determine whether human medulloblastoma derived cell lines express interleukin-4 receptor and whether interleukin-4 receptor expression is accompanied by sensitivity to cpIL4-PE . Medulloblastoma cell lines express interleukin-4 receptor at the protein and mRNA levels as determined by binding, indirect immunofluorescence and RT--PCR studies . These cells expressed IL-4Ralpha (also known as IL-4Rbeta) and IL-13Ralpha1 (also known as IL-13Ralpha') chains, however common gamma(c), a component of the interleukin-4 receptor system in immune cells was not detected . Consistent with the expression of IL-4R, cpIL4-PE was found to be highly and specifically cytotoxic to four of five medulloblastoma cell lines . Susceptibility of medulloblastoma cell lines to cpIL4-PE seemed to correlate closely to the functional IL-4 binding sites in general as demonstrated by 125I-IL-4 binding, but did not seem to correlate with mRNA or cell surface immunoreactive receptor protein expression . The sensitivity of medulloblastoma cells to cpIL4-PE could be eliminated by concurrent incubation with IL-4 or IL-13, but not with IL-2 . None of these cell lines showed any change in proliferation upon treatment with exogenous IL-4 . These studies establish the interleukin-4 receptor as a medulloblastoma-associated target for possible tumour-directed cancer therapy . Further studies are warranted to investigate interleukin-4 receptor expression in primary medulloblastoma tumours and sensitivity to cpIL-4PE in vitro and in vivo . Plant J, 2002 Jan, 29(2), 131 - 40 Esa1, an Arabidopsis mutant with enhanced susceptibility to a range of necrotrophic fungal pathogens, shows a distorted induction of defense responses by reactive oxygen generating compounds; Tierens KF et al.; An Arabidopsis thaliana mutant, esa1, that shows enhanced susceptibility to the necrotrophic pathogens Alternaria brassicicola, Botrytis cinerea and Plectosphaerella cucumerina, but has wild-type levels of resistance to the biotrophic pathogens Pseudomonas syringae pv . tomato and Peronospora parasitica . The enhanced susceptibility towards necrotrophic pathogens correlated with a delayed induction of phytoalexin accumulation and delayed induction of the plant defensin gene PDF1.2 upon inoculation with pathogens . Two reactive oxygen generating compounds, paraquat and acifluorfen, were found to cause induction of both phytoalexin accumulation and PDF1.2 expression in wild-type plants, but this induction was almost completely abolished in esa1 . This finding suggests that esa1 may somehow be involved in transduction of signals generated by reactive oxygen species. Cancer Immunol Immunother, 2002 Feb, 50(12), 691 - 700 Epub 2001 Dec 07. Apoptotic pathways of cell death induced by an interleukin-13 receptor-targeted recombinant cytotoxin in head and neck cancer cells; Kawakami M et al.; Interleukin 13 receptor (IL-13R)-targeted cytotoxin, IL13-PE38QQR, composed of IL-13 and a mutated form of Pseudomonas exotoxin (PE), is found to be highly and specifically cytotoxic to human solid cancer cell lines . However, the mechanism of tumor cell death mediated by IL-13 toxin is still not known . To elucidate the mechanism, we utilized four head and neck cancer cell lines (SCC-25, HN12, KCCT873, and YCUM911), which express high levels of IL-13R, and IL-13 toxin is highly cytotoxic to these cells . We observed chromatin condensation and DNA fragmentation, indicating apoptotic cell death, after treatment with IL-13 toxin, as determined by bis-benzimide staining and DNA ladder assays . However, IL-13 did not induce cell death . Flow cytometric analysis suggested that these cancer cell lines increased the sub-G1/G0 phase DNA population in a dose- and time-dependent manner (ranged between 10 and 30%) after treatment with IL-13 toxin . By Western blot analysis, cleavage of caspase-3 and PARP was observed after treatment with a high concentration of IL-13 toxin, also suggesting apoptotic cell death . In addition, the results of immunofluorescence and RT-PCR assays showed that the apoptosis-regulator, Bcl-2 was downregulated after treatment with IL-13 toxin, while Bax was upregulated . Moreover, significant nitrite production was detected in the HN12 cell line after treatment with IL-13 toxin for 48--96 h . Taken together, our results suggest that IL-13 toxin-induced cytotoxicity is at least partially mediated by the apoptosis and nitric oxide pathways . This information may be useful in developing specific approaches where apoptotic bodies from tumor cells may be used to pulse antigen-presenting cells for immunotherapy of cancer. Carbohydr Res, 2002 Mar 1, 337(5), 467 - 71 O-Specific chain structure from the lipopolysaccharide fraction of Pseudomonas reactans: a pathogen of the cultivated mushrooms; Molinaro A et al.; An O-specific polysaccharide containing 2-acetamidino-2-deoxy-beta-D-glucopyranose (Glcp2Am), 2,4-diacetamido-2,4,6-trideoxy-beta-D-glucopyranose (QuipNAc4NAc, bacillosamine) and 2,4-di-(N-acetyl-L-alanylamino)-2,4,6-trideoxy-beta-D-glucopyranose (QuipNAlaAc4NAlaAc) was isolated from the phenol-soluble lipopolysaccharide fraction of the mushroom-associated bacterium Pseudomonas reactans . The structure, determined by means of chemical analysis and 1D and 2D NMR spectroscopy, showed a linear trisaccharide-repeating unit, as shown below:-->3)-beta-D-QuipNAlaAc4NAlaAc-(1-->3)-alpha-D-Glcp2Am-(1-->3)-alpha-D-QuipNAc4NAc(1-->To our knowledge, this is the first complete O-chain structure reported for the lipopolysaccharide of a mushroom-associated bacterium. Diagn Microbiol Infect Dis, 2002 Feb, 42(2), 141 - 3 Leg ulcer due to Pseudomanas luteola in a patient with sickle cell disease; Tsakris A et al.; Pseudomonas luteola is rarely implicated in clinical infections, usually in association with indwelling catheters or prostheses . This report describes the first case of deteriorated leg ulcer caused by a multiply antibiotic resistant P . luteola strain in a patient with homozygous sickle cell disease. JOP, 2000 Nov, 1(4), 208 - 10 Microvascular complications in cystic fibrosis-related diabetes mellitus: a case report; Scott AI et al.; CONTEXT, The prevalence of cystic fibrosis-related diabetes mellitus is increasing and is associated with increased survival from cystic fibrosis . CASE REPORT, This study describes a case of the premature onset of disabling and widespread microvascular complications resulting from cystic fibrosis-related diabetes mellitus . Previously asymptomatic retinopathy was diagnosed on recognition of diabetic nephropathy . CONCLUSIONS, The treatment of pulmonary exacerbations has become more complex due to the nephrotoxic potential of intravenous aminoglycoside drugs which are frequently used to control chronic Pseudomonas infection in cystic fibrosis. Plant Mol Biol, 2002 Feb 1, 48(3), 267 - 76 Infection of Arabidopsis with a necrotrophic pathogen, Botrytis cinerea, elicits various defense responses but does not induce systemic acquired resistance (SAR); Govrin EM et al.; Botrytis cinerea is a non-specific necrotrophic pathogen that attacks more than 200 plant species . In contrast to biotrophs, the necrotrophs obtain their nutrients by first killing the host cells . Many studies have shown that infection of plants by necrosis-causing pathogens induces a systemic acquired resistance (SAR), which provides protection against successive infections by a range of pathogenic organisms . We analyzed the role of SAR in B . cinerea infection of Arabidopsis . We show that although B . cinerea induced necrotic lesions and camalexin biosynthesis, it did not induce SAR-mediated protection against virulent strains of