Appl Microbiol Biotechnol . 2005 Jan 15; {Epub ahead of print} Cloning and expression of a trehalose synthase from Pseudomonas stutzeri CJ38 in Escherichia coli for the production of trehalose; Lee JH et al.; A novel strain was isolated, Pseudomonas stutzeri CJ38, that enabled direct transformation of maltose to trehalose . In comparison with others reported to date, CJ38 provided a novel trehalose synthase (TSase) without any byproduct, including glucose . Activity analysis, using either maltose or trehalose as a substrate, showed a reversible reaction . There was also no detectable activity of related enzymes with liquid starch and maltooligosaccharides as substrates . Using a malPQ-negative host and MacConkey medium, the TSase gene was cloned in Escherichia coli from CJ38 . The resulting sequence contained an open reading frame consisted of 689 amino acids with a calculated molecular mass of 76 kDa . A search for related sequences in various gene and protein data banks revealed a novel family of enzymes that was predicted putatively as a glycosidase or TSase family, with no biochemical evidence . The recombinant enzyme exhibited a high activity toward the substrate maltose, about 50-fold higher than the parent strain and resulted in a high conversion yield (72%) at a relatively high substrate concentration (20%) . These results provided the possibility that the strain was effectively used as a potential biocatalyst for the production of trehalose from maltose in a one-step reaction. J Microbiol, 2004 Dec, 42(4), 365 - 8 Occurrence of the strA-strB streptomycin resistance genes in Pseudomonas species isolated from kiwifruit plants; Han HS et al.; The occurrence of strA-strB streptomycin-resistance genes within transposon Tn5393 was examined in Pseudomonas syringae pv . actinidiae, P . syringae pv . syringae, and P . marginalis, isolated from kiwifruit plants in Korea and Japan . PCR amplification with primers specific to strA-strB revealed that three of the tested Pseudomonas species harbored these genes for a streptomycin-resistance determinant . Tn5393, containing strA-strB, was also identified with PCR primers designed to amplify parts of tnpA, res, and tnpR . No IS elements were detected within tnpR, nor were they found in the intergenic region between tnpR and strA . Nucleotide sequence analysis indicated that the strA sequence of P . syringae pv . actinidiae contained a single nucleotide alteration at position 593 (CAA-->CGA), as compared to Tn5393a in P . syringae pv . syringae . This resulted in an amino acid change, from Gln to Arg. Int J Mol Med, 2005 Feb, 15(2), 305 - 13 Recombinant anti-EGFR immunotoxin 425(scFv)-ETA' demonstrates anti-tumor activity against disseminated human pancreatic cancer in nude mice; Bruell D et al.; Pancreatic carcinoma is the fifth leading cause of cancer-related deaths in North America and Europe . Major reasons for the high mortality rate include the inability to detect pancreatic cancer at an early stage, extensive local invasion, and early formation of lymphatic and hematogenous metastases . Consequently, novel and effective therapies need to be developed urgently in order to improve the outcome of patients . Since overexpression of the epidermal growth factor receptor (EGFR) in pancreatic tumors correlates with advanced clinical staging, increased tumor size and reduced patient survival, this receptor represents an appropriate target for immunotherapy . We recently generated the recombinant immunotoxin 425(scFv)-ETA' by genetically fusing the anti-EGFR single chain variable fragment 425(scFv) to a truncated version of Pseudomonas aeroginosa exotoxin A (ETA') . The 425(scFv)-ETA' fusion protein was functionally expressed in the periplasmic space of Escherichia coli and was purified using a combination of metal-ion affinity and anion exchange chromatography . The protein showed specific binding to and toxicity against the EGFR-positive, metastatic pancreatic carcinoma cell line L3.6pl, but not to control cell systems . We report the anti-tumor activity of this recombinant immunotoxin in a disseminated human pancreatic cancer nude mouse model . After intravenous (i.v.) injection of L3.6pl cells into immunodeficient nude mice, both single (20 microg on day 1 after challenge) and repeated (10 microg on days 1, 2, 3 and 4 after tumor cell injection) i.v . administration of 425(scFv)-ETA' resulted in a significant reduction in the average number of lung metastases from 56.25 per animal in the control groups to 0.875 per animal (single injection) and 0.286 per animal (repeated injection), respectively, in the experimental groups . In summary, this is the first report showing an in vivo anti-tumor effect caused by the recombinant immunotoxin 425(scFv)-ETA' against disseminated growing metastatic human pancreatic carcinoma cells . Our data suggest that EGFR-specific antibody toxins could be suitable for further clinical investigation in the development of therapies for pancreatic carcinoma. Can J Microbiol, 2004 Sep, 50(9), 691 - 696 Sporocarps of Pisolithus albus as an ecological niche for fluorescent pseudomonads involved in Acacia mangium Wild &ndash; Pisolithus albus ectomycorrhizal symbiosis; Duponnois R et al.; Fresh sporocarps and root and soil samples were collected under a monospecific forest plantation of Acacia mangium in Dagana in Northern Senegal and checked for the presence of fluorescent pseudomonads . No bacteria were detected except from sporocarps collected with adhering soil and hyphal strands . Pisolithus sporocarps were dried at 30 degrees C for 2 weeks, ground, passed through a 2-mm sieve and mixed together . This dry sporocarp powder (DSP) was used to inoculate and form mycorrhizas on A . mangium seedlings in a glasshouse experiment . After 3 months culture, plant growth was increased in the DSP treatment but no ectomycorrhizas were present on the A . mangium root systems; however fluorescent pseudomonads were recorded in the cultural soil . The stimulatory effects on the plant growth were maintained for 6 months . However, fluorescent pseudomonads were no longer detected and 35% of the short roots were ectomycorrhizal . Some of the fluorescent pseudomonad isolates detected after 3 months stimulated the radial fungal growth in axenic conditions . These observations suggest that these bacteria are closely associated with the Pisolithus fructifications and could interact with the ectomycorrhizal symbiosis establishment. Appl Environ Microbiol, 2005 Jan, 71(1), 569 - 73 Mutation of a LysR-Type Regulator of Antifungal Activity Results in a Growth Advantage in Stationary Phase Phenotype in Pseudomonas aureofaciens PA147-2; Silby MW et al.; The growth advantage in stationary phase (GASP) phenotype was shown to be present in two mutants lacking the antifungal phenotype (Af(-) mutants) of Pseudomonas aureofaciens PA147-2 . Complementation demonstrated a correlation between GASP and the antifungal defect in one strain but not in the second . Sequence analysis revealed the Af(-) GASP strain had a mutation in a gene (finR) encoding a LysR-type regulator . Antifungal-minus mutants arose in starved cultures, and those aged cultures had increased fitness . Taken together, the results show that there are at least two paths to the GASP phenotype in P . aureofaciens, one of which results in a concomitant loss of the antifungal phenotype. Carbohydr Res, 2005 Feb 7, 340(2), 319 - 23 Enzymatic procedures in the preparation of regioprotected d-fructose derivatives; D'Antona N et al.; A combination of different lipases from Pseudomonas cepacia, Candida antarctica B, Candida rugosa and Mucor miehei, aided the regioesterification of the free fructose allowing the synthesis of 1,6-di-O-acetyl-d-fructofuranose, 1,4,6-tri-O-acetyl-d-fructofuranose, 1,6-di-O-acetyl-4-O-benzoyl-d-fructofuranose and 1,6-di-O-benzoyl-d-fructofuranose . Using C . antarctica B and C . rugosa lipases the alcoholysis of fructose peracetate (alpha, beta-form) has furnished 1,2,3,4-tetra-O-acetyl-alpha-d-fructofuranose and 2,3,4,6-tetra-O-acetyl-beta-d-fructofuranose . 1,4,6-Tri-O-acetyl-d-fructofuranose was successfully employed to produce a rare ketohexose, namely d-psicose. Biomacromolecules, 2005 Jan 10, 6(1), 99 - 104 Synergistic Effects of Glu130Asp Substitution in the Type II Polyhydroxyalkanoate (PHA) Synthase: Enhancement of PHA Production and Alteration of Polymer Molecular Weight; Matsumoto K et al.; In vitro evolution of the polyhydroxyalkanoate (PHA) synthase gene from Pseudomonas sp . 61-3 (phaC1(Ps)) has been performed to generate highly active enzymes . In this study, a positive mutant of PHA synthase, Glu130Asp (E130D), was characterized in detail in vivo and in vitro . Recombinant Escherichia coli strain JM109 harboring the E130D mutant gene accumulated 10-fold higher (1.0 wt %) poly(3-hydroxybutyrate) {P(3HB)} from glucose, compared to recombinant E . coli harboring the wild-type PHA synthase gene (0.1 wt %) . Recombinant E . coli strain LS5218 harboring the E130D PHA synthase gene grown on dodecanoate produced more poly(3HB-co-3-hydroxyalkanoate) {P(3HB-co-3HA)} (20 wt %) copolymer than an LS5218 strain harboring the wild-type PHA synthase gene (13 wt %) . The E130D mutation also resulted in the production of copolymer with a slight increase in 3HB composition, compared to copolymer produced by the wild-type PHA synthase . In vitro enzyme activities of the E130D PHA synthase toward various 3-hydroxyacyl-CoAs (4-10 carbons in length) were all higher than those of the wild-type enzyme . The combination of the E130D mutation with other beneficial mutations, such as Ser325Thr and Gln481Lys, exhibited a synergistic effect on in vivo PHA production and in vitro enzyme activity . Interestingly, gel-permeation chromatography analysis revealed that the E130D mutation also had a synergistic effect on the molecular weight of polymers produced in vivo. J Biol Chem . 2005 Jan 5; {Epub ahead of print} Gene trap mutagenesis-based forward genetic approach reveals that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor-2; Nobukuni Y et al.; OVCA1 is a tumor suppressor identified by positional cloning from chromosome 17p13.3, a hot spot for chromosomal aberration in breast and ovarian cancers . It has been shown that expression of OVCA1 is reduced in some tumors and that it regulates cell proliferation, embryonic development, and tumorigenesis . However, the biochemical function of OVCA1 has remained unknown . Recently, we isolated a novel mutant resistant to diphtheria toxin (DT) and Pseudomonas exotoxin A (ETA) from the gene trap insertional mutants library of Chinese hamster ovary (CHO) cells . In this mutant, the OVCA1 gene was disrupted by gene trap mutagenesis, and this disruption well correlated with the toxin resistant-phenotype . We demonstrated direct evidence that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor 2 (EF-2), the target of bacterial ADP-ribosylating toxins . A functional genetic approach utilizing the random gene trap mutants library of mammalian cells should become a useful strategy to identify the genes responsible for specific phenotypes. Plant J, 2005 Jan, 41(2), 304 - 18 An Arabidopsis NPR1-like gene, NPR4, is required for disease resistance; Liu G et al.; Summary The Arabidopsis genome contains six NPR1-related genes . Given the pivotal role played by NPR1 in controlling salicylic acid (SA)-mediated gene expression and disease resistance, functional characterization of other family members appears to be justified . Reverse genetics was used to analyze the role of one NPR1-like gene, which we called NPR4 . The NPR4 protein shares 36% identity with NPR1 and interacts with the same spectrum of TGA transcription factors in yeast two-hybrid assays . Plants with T-DNA insertions in NPR4 are more susceptible to the virulent bacterial pathogen Pseudomonas syringe pv . tomato DC3000 . This phenotype is complemented by expression of the wild type NPR4 coding region . As determined by the parasite reproduction, the npr4-1 mutant is more susceptible to the fungal pathogen Erysiphe cichoracearum, but does not differ markedly from wild type in its interaction with virulent and avirulent strains of the oomycete Peronospora parasitica . In leaves of wild-type plants, NPR4 mRNA levels increase following pathogen challenge or SA treatment, and decrease rapidly following methyl jasmonic acid (MeJA) treatment . Transcripts of the pathogenesis-related (PR) genes PR-1, PR-2, and PR-5 are only marginally reduced in the npr4-1 mutant following pathogen challenge or SA treatment . This reduction of PR gene expression is more pronounced when leaves are challenged with the bacterial pathogen following SA treatment . Expression of the jasmonic acid-dependent pathway marker gene PDF1.2 is compromised in npr4-1 leaves following application of MeJA or a combination of SA and MeJA . These results indicate that NPR4 is required for basal defense against pathogens, and that it may be implicated in the cross-talk between the SA- and JA-dependent signaling pathways. Otolaryngol Head Neck Surg, 2005 Jan, 132(1), 25 - 9 Hearing loss and extent of labyrinthine injury in Pseudomonas otitis media; Malaty J et al.; Objectives To determine if the severity of hearing loss due to semicircular canal transection (SCT) in Pseudomonas otitis media (POM) is a function of the extent of the labyrinthine injury . Methods POM was induced bilaterally in 45 guinea pigs . After 2 to 4 days, SCT was performed in one 1 on the horizontal canal, the superior canal, or both the horizontal and the superior canals . Auditory-evoked brainstem responses were measured before and after SCT . Results POM was induced bilaterally in all animals . Elevation of click thresholds was found in almost all SCT ears of all 3 groups; however, there were no significant differences between groups . Comparing the SCT ear to the contralateral, control ear, mean hearing losses in the horizontal, superior, and horizontal + superior SCT groups were 23 dB, 27 dB, and 24 dB, respectively . Conclusions Hearing loss does not correlate with extent of labyrinthine injury in POM in the guinea pig . EBM rating: B-2. Microbiology, 2005 Jan, 151(Pt 1), 269 - 80 Type III secretion chaperones ShcS1 and ShcO1 from Pseudomonas syringae pv . tomato DC3000 bind more than one effector; Kabisch U et al.; The hrp-type III secretion (TTS) system is a key pathogenicity factor of the plant pathogen Pseudomonas syringae pv . tomato DC3000 that translocates effector proteins into the cytosol of the eukaryotic host cell . The translocation of a subset of effectors is dependent on specific chaperones . In this study an operon encoding a TTS chaperone (ShcS1) and the truncated effector HopS1' was characterized . Yeast two-hybrid analysis and pull-down assays demonstrated that these proteins interact . Using protein fusions to AvrRpt2 it was shown that ShcS1 facilitates the translocation of HopS1', suggesting that ShcS1 is a TTS chaperone for HopS1' and that amino acids 1 to 118 of HopS1' are required for translocation . P . syringae pv . tomato DC3000 carries two shcS1 homologues, shcO1 and shcS2, which are located in different operons, and both operons include additional putative effector genes . Transcomplementation experiments showed that ShcS1 and ShcO1, but not ShcS2, can facilitate the translocation of HopS1' : : AvrRpt2 . To characterize the specificities of the putative chaperones, yeast two-hybrid interaction studies were performed between the three chaperones and putative target effectors . These experiments showed that both ShcS1 and ShcO1 bind to two different effectors, HopS1' and HopO1-1, that share only 16 % amino acid sequence identity . Using gel filtration it was shown that ShcS1 forms homodimers, and this was confirmed by yeast two-hybrid experiments . In addition, ShcS1 is also able to form heterodimers with ShcO1 . These data demonstrate that ShcS1 and ShcO1 are exceptional class IA TTS chaperones because they can bind more than one target effector. J Biochem (Tokyo), 2004 Nov, 136(5), 617 - 27 Sequencing and Expression of the L-Phenylalanine Oxidase Gene from Pseudomonas sp . P-501 . Proteolytic Activation of the Proenzyme; Suzuki H et al.; The nucleotide sequence encoding l-phenylalanine oxidase (deaminating and decarboxylating) (PAO) from Pseudomonas sp . P-501 was determined . The open reading frame is arranged in the order of prosequence, alpha subunit, dipeptide and beta subunit from the 5'- to 3'-end . Expression of the gene in Escherichia coli showed that without the prosequence, PAO is produced in small quantity as a soluble form with no visible absorption, but with the prosequence (proPAO), PAO is highly expressed and yellow . The purified proPAO contained one mol of FAD per mol of proPAO polypeptide, but had no catalytic activity . Treatment of proPAO with a mixture of Pronase and trypsin converted the noncatalytic proPAO to the catalytic form, and the Pronase-trypsin-treated proPAO showed kinetic and spectral properties comparable to the native enzyme . These results suggest that in Pseudomonas, PAO is expressed as a proenzyme that is processed by proteolysis to the active form. J Biol Chem . 2005 Jan 4; {Epub ahead of print} Nitrile pathway involving Acyl-CoA synthetase: Overall metabolic gene organization, and purification and characterization of the enzyme; Hashimoto Y et al.; Two open reading frames (nhpS and acsA) were identified immediately downstream of the previously described Pseudomonas chlororaphis B23 nitrile hydratase (NHase) gene cluster (encoding aldoxime dehydratase, amidase, the two NHase subunits, and an uncharacterized protein) . The amino acid sequence deduced from acsA shows similarity to that of acyl-CoA synthetase . The acsA gene product expressed in Escherichia coli showed acyl-CoA synthetase activity toward butyric acid and CoA as substrates, butyryl-CoA being synthesized . From the E . coli transformant, AcsA was purified to homogeneity and characterized . The quality of the recombinant protein was verified by the N-terminal amino acid sequence and the results of MALDI-TOF mass spectrometry . The apparent Km values for butyric acid, CoA, and ATP were 0.32 0.04, 0.37 0.02, and 0.22 0.02 mM, respectively . AcsA was shown to be a short-chain acyl-CoA synthetase, according to the catalytic efficiencies (kcat/Km) for various acids . The substrate specificity of AcsA was similar to those of aldoxime dehydratase, NHase, and amidase, the genes of which co-exist in the same orientation in the gene cluster . P . chlororaphis B23 grew when cultured in a medium containing butyraldoxime as the sole carbon and nitrogen source . The activities of aldoxime dehydratase, NHase, and amidase were detected together with that of acyl-CoA synthetase under the culture conditions used . Moreover, on culture in a medium containing butyric acid as the sole carbon source, acyl-CoA synthetase activity was also detected . Together with the adjacent locations of the aldoxime dehydratase, NHase, amidase, and acyl-CoA synthetase genes, these findings suggest that the four enzymes are sequentially correlated with one another in vivo to utilize butyraldoxime as a carbon and nitrogen source . This is the first report of an overall nitrile pathway (aldoxime -> nitrile -> amide -> acid -> acyl-CoA) comprising these enzymes. J Bacteriol, 2005 Jan, 187(2), 593 - 600 Molecular Nature of Spontaneous Modifications in gacS Which Cause Colony Phase Variation in Pseudomonas sp . Strain PCL1171; van den Broek D et al.; Pseudomonas sp . strain PCL1171 displays colony phase variation between opaque phase I and translucent phase II colonies, thereby regulating the production of secondary metabolites and exoenzymes . Complementation and sequence analysis of 26 phase II mutants and of 13 wild-type phase II sectors growing out of phase I colonies showed that in all these cases the phase II phenotype is caused by spontaneous mutations in gacA or/and gacS . Mutation of gac reduced both the length of the lag phase and the generation time . Isolation and sequencing of the gacS genes from the phase II bacteria revealed one insertion as well as several random point mutations, deletions, and DNA rearrangements . Most phase II colonies reverted with a high frequency, resulting in wild-type gacA and gacS genes and a phase I phenotype . Some phase II bacteria retained the phase II phenotype but changed genotypically as a result of (re)introduction of mutations in either gacA or gacS . The reversion of gacA or gacS to the wild type was not affected by mutation of recA and recB . We conclude that in Pseudomonas sp . strain PCL1171, mutations in gacA and gacS are the basis for phase variation from phase I to phase II colonies and that, since these mutations are efficiently removed, mutations in gac result in dynamic switches between the "wild-type" population and the subpopulations harboring spontaneous mutations in gacA and or gacS, thereby enabling both populations to be maintained. Arch Biochem Biophys, 2005 Feb 1, 434(1), 67 - 74 NahR: effects of replacements at Asn 169 and Arg 248 on promoter binding and inducer recognition; Park HH et al.; NahR, a member of the LysR regulator family, is a positive transcriptional regulator for genes of the naphthalene degradation pathway in Pseudomonas sp . To study NahR binding properties, five single and six double mutants were made at residues 169 and/or 248, which are located in the central inducer recognition domain and the C-terminal multimerization domain of the protein, respectively . The effects of these mutations were examined by monitoring the expression of a firefly luciferase (luc) reporter gene under the control of NahR . We found that all mutants responded to induction by both salicylate and benzoate, whereas the wild-type NahR responded only to salicylate . Mutants N169E, N169E/R248C, and N169E/R248K showed low basal activities with high-level inducer responses, whereas mutant N169D/R248K showed high basal activity with inducer-independent responses . A gel retardation assay demonstrated that the different basal activities might be related to altered binding affinities of the NahR mutants to the Psal promoter . Together, these data suggest that NahR residues 169 and 248 might be involved in DNA binding as well as inducer recognition/binding. Blood . 2004 Dec 30; {Epub ahead of print} Hodgkin's lymphoma therapy with interleukin-4 receptor-directed cytotoxin in an infiltrating animal model; Kawakami M et al.; Hodgkin's lymphoma represents unique clinicopathological features because Hodgkin and Reed-Sternberg (H-RS) cells produce a variety of cytokines, express a variety of cytokine receptors, and are surrounded by numerous non-malignant immunoreactive cells . We found that receptors for interleukin-4 (IL-4R) are highly expressed in H-RS cells . To target IL-4R, we utilized a recombinant protein fusing circularly permuted human IL-4 and Pseudomonas exotoxin termed IL4(38-37)-PE38KDEL or IL-4 cytotoxin . The cytotoxic effect of IL-4 cytotoxin on H-RS cell lines was determined to be moderate to high in vitro . We developed an infiltrating model of Hodgkin's disease by injecting an adherent population of HD-MyZ cells subcutaneously into the flanks of beige/nude/X-linked immunodeficient mice . The animal model exhibited spontaneous metastasis of H-RS cells to lymph nodes as well as dissemination to vital organs including the lungs . Intraperitoneal or intratumoral treatment of these mice with IL-4 cytotoxin resulted in regression of the primary tumor mass and a decrease in the incidence of lymph node metastasis . Mice injected with HD-MyZ cells demonstrated 203% prolonged survival (mean survival, 63 days) compared to control (mean survival, 31 days) when they received systemic IL-4 cytotoxin treatment . Because a variety of H-RS cell lines express receptors for IL-4, IL-4 cytotoxin may be a unique agent for the treatment of Hodgkin's lymphoma. DNA Seq, 2004 Oct-Dec, 15(5-6), 338 - 43 Lateral Gene Transfer in the Deep Sea of Mariana Trench: Identification of nar Gene Cluster Encoding Membrane-bound Nitrate Reductase from Pseudomonas sp . Strain MT-1; Tamegai H et al.; The genes encoding membrane-bound nitrate reductase and its locus from Pseudomonas sp . strain MT-1, which is isolated from the sediment of Mariana Trench, were identified . To some extent, the gene organization in the cluster was different from those of other Pseudomonads . Quite interestingly, two genes encoding putative nitrate transporter (narK and narM) showed higher homologies to counterparts of organisms belonging to other genera than those of Pseudomonads . Especially, narM showed no significant homology to the genes for nitrate transporter of Pseudomonads, and was homologous to those of some marine bacteria . Further, arrangements of NarL- and Fnr-binding motifs in the cluster were different from those of P . stutzeri, closely related strain with MT-1 . These observations clearly indicated that lateral transfer of genes in nar gene cluster had occurred in deep sea, and it may contribute to bacterial adaptation to environment of there. FEMS Microbiol Lett, 2005 Jan 15, 242(2), 249 - 56 Antagonistic activity among 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp; Validov S et al.; Strains of fluorescent Pseudomonas spp . that produce 2,4-diacetylphloroglucinol (2,4-DAPG) differ in their ability to colonize roots . In this study, we screened 47 2,4-DAPG-producing strains representing17 distinct genotypes for antagonistic activity associated with the production of bacteriocins . Upon induction, over 70% of the strains inhibited the growth of other isolates in vitro . Greenhouse assays indicated that populations of sensitive strains in wheat rhizosphere soil declined more rapidly in the presence of antagonists than when introduced alone . Antagonism can influence the ability of biocontrol agents to establish and maintain effective population densities in situ. J Cataract Refract Surg, 2004 Dec, 30(12), 2569 - 73 Perioperative complications of intraocular lens exchange in patients with opacified Aqua-Sense lenses; Dagres E et al.; PURPOSE: To evaluate the perioperative complications of intraocular lens (IOL) exchange in 25 eyes of 22 patients with opacified Aqua-Sense(R) IOLs (Ophthalmic Innovations International) . SETTING: Department of Ophthalmology, University Hospital Aintree, Liverpool, United Kingdom . METHODS: The study comprised 22 patients (25 eyes) who had previous phacoemulsification and implantation of Aqua-Sense single-piece hydrophilic acrylic IOLs in the capsular bag and developed severe late opacification of the IOL . All patients reported glare and deterioration in vision . The IOLs were explanted and replaced with new lenses . The perioperative complications were evaluated . The best corrected visual acuity (BCVA) before and after surgery was compared . RESULTS: In 24 eyes, the opacification was complete, involving the optic, haptics, and substance of the IOLs . Uneventful IOL exchange and placement of a new IOL in the bag was achieved in 13 eyes (52%) . Complications occurred in the remaining 12 eyes (48%) . Ten eyes (40%) developed zonular dehiscence, 4 (16%) of which were managed with anterior chamber IOL implantation . One eye (4%) developed posterior capsule rupture and 1 eye (4%), posterior capsule rupture and zonular dehiscence . The cornea decompensated in 2 eyes (8%) . One eye (4%) developed Pseudomonas keratitis . The mean BCVA (decimal scale) before and after IOL exchange was 0.57 +/- 0.24 and 0.60 +/- 0.28, respectively . There was no significant difference in visual acuity between before and after IOL exchange (P=.782, paired t test) . CONCLUSIONS: Explantation of Aqua-Sense IOLs was challenging because of the tight adherence of the optic and haptics to the capsule . Long-term follow-up of patients with Aqua-Sense IOLs should be maintained. Lett Appl Microbiol, 2005, 40(1), 30 - 6 Biodesulfurization using Pseudomonas delafieldii in magnetic polyvinyl alcohol beads; Guobin S et al.; Abstract s . guobin, x . jianmin, g . chen, l . huizhou and c . jiayong . 2004.Aims: To immobilize Pseudomonas delafieldii R-8 cells in magnetic polyvinyl alcohol (PVA) beads for biodesulfurization . Methods and Results: Magnetic PVA beads were prepared by a freezing-thawing technique under liquid nitrogen . The beads have distinct super-paramagnetic properties and their saturation magnetization is 8.02 emu g(-1) . The desulfurization rate of the immobilized cells could reach 40.2 mmol kg(-1) h(-1) . Desulfurization patterns of dibenzothiophene in model oil with the immobilized and free cells were represented by the Michaelis-Menten equation . The Michaelis constant for both immobilized and free cells was 1.3 mmol l(-1) . Conclusions: The cells immobilized in magnetic PVA beads could be stably stored and be repeatedly used over 12 times for biodesulfurization . The immobilized cells could be easily separated by magnetic field . Significance and Impact of the Study: Magnetic PVA beads are easy to prepare . The immobilization process in the paper is to increase the efficiency of cells and to decrease the cost of operations. Bioresour Technol, 2005 May, 96(7), 769 - 77 Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil; Noureddini H et al.; Enzymatic transesterification of soybean oil with methanol and ethanol was studied . Of the nine lipases that were tested in the initial screening, lipase PS from Pseudomonas cepacia resulted in the highest yield of alkyl esters . Lipase from Pseudomonas cepacia was further investigated in immobilized form within a chemically inert, hydrophobic sol-gel support . The gel-entrapped lipase was prepared by polycondensation of hydrolyzed tetramethoxysilane and iso-butyltrimethoxysilane . Using the immobilized lipase PS, the effects of water and alcohol concentration, enzyme loading, enzyme thermal stability, and temperature in the transesterification reaction were investigated . The optimal conditions for processing 10g of soybean oil were: 35 degrees C, 1:7.5 oil/methanol molar ratio, 0.5g water and 475mg lipase for the reactions with methanol, and 35 degrees C, 1:15.2 oil/ethanol molar ratio, 0.3g water, 475mg lipase for the reactions with ethanol . Subject to the optimal conditions, methyl and ethyl esters formation of 67 and 65mol% in 1h of reaction were obtained for the immobilized enzyme reactions . Upon the reaction with the immobilized lipase, the triglycerides reached negligible levels after the first 30min of the reaction and the immobilized lipase was consistently more active than the free enzyme . The immobilized lipase also proved to be stable and lost little activity when was subjected to repeated uses. Plant Mol Biol, 2004 Jul, 55(4), 607 - 18 Overexpression of the AP2/EREBP transcription factor OPBP1 enhances disease resistance and salt tolerance in tobacco; Guo ZJ et al.; Osmotin promoter binding protein 1 (OPBP1), an AP2/EREBP-like transcription factor of tobacco (Nicotiana tabacum), was isolated using a yeast one-hybrid system . RNA gel blot analysis indicated that expression of the OPBP1 gene was induced by elicitor cryptogein, NaCl, ethephon, methyl jasmonate, as well as cycloheximide . Transient expression analysis using an OPBP1-eGFP fusion gene in onion epidermal cells revealed that the OPBP1 protein was targeted to the nuclear . Further, electrophoretic mobility shift assays demonstrated that the recombinant OPBP1 protein could bind to an oligonucleotide containing the GCC-box cis element . Transgenic tobacco plants with an over expression of the OPBP1 gene accumulated high levels of PR-1a and PR-5d genes and exhibited enhanced resistance to infection by Pseudomonas syringae pv tabaci and Phytophthora parasitica var nicotianae pathogens . They also exhibited increased tolerance to salt stress . These results suggest that OPBP1 might be a transcriptional regulator capable of regulating expression in sets of stress-related genes. Plant Mol Biol, 2004 May, 55(1), 61 - 81 The ethylene-responsive factor like protein 1 (CaERFLP1) of hot pepper (Capsicum annuum L.) interacts in vitro with both GCC and DRE/CRT sequences with different binding affinities: possible biological roles of CaERFLP1 in response to pathogen infection and high salinity conditions in transgenic tobacco plants; Lee JH et al.; From a pathogen-inoculated hot pepper (Capsicum annuum L . cv . Pukang) leaf EST, we identified a cDNA clone, pCaERFLP1, encoding a putative transcription factor that contains a single ERF/AP2 DNA binding domain . CaERFLP1 was most closely related to tomato LeERF2 (73%), both of which belong to the novel ERF class IV typified by the N-terminal MCGGAIL signature sequence, while it had a limited sequence identity (25-30%) with Arabidopsis AtERFs and tobacco NtERFs . Quantitative gel retardation assays revealed that bacterially expressed full-length CaERFLP1 was able to form a specific complex with both the GCC box and DRE/CRT motif, with its binding affinity for GCC being stronger than for DRE/CRT . When fused to the GAL4 DNA binding domain, the N-terminal CaERFLP1(1-37) and C-terminal CaERFLP1(198-264) mutant polypeptides could function individually as transactivators in yeast . This suggests that two separate domains of CaERFLP1 may play distinct roles in transcription activation . In particle co-bombardment experiments, CaERFLP1 activated the transcription of reporter genes containing the 4X{GCC} element in tobacco cells . In hot pepper plants, the steady-state level of CaERFLP1 mRNA was markedly induced by multiple environmental factors, such as pathogen infection, ethylene, mechanical wounding and high salinity . Furthermore, ectopic expression of CaERFLP1 in transgenic tobacco plants resulted in partially improved tolerance against the bacterial pathogen Pseudomonas syringae and salt stress (100 mM NaCl) . Consistently, various defense-related genes, including GCC box-containing PR genes and the DRE/CRT-containing LTI45 (ERD10) gene, were constitutively expressed in 35S::CaERFLP1 tobacco plants . Thus, it appears that CaERFLP1 is functional in tobacco cells, where it induces the transactivation of some GCC- and DRE/CRT-genes to trigger a subset of stress response . Here, the possible biological role(s) of CaERFLP1 is discussed, especially with regard to the possibility that CaERFLP1 has multiple functions in the regulation of GCC- and DRE/CRT-mediated gene expression in hot pepper plants. Plant Mol Biol, 2004 Apr, 54(6), 817 - 35 Crosstalk and differential response to abiotic and biotic stressors reflected at the transcriptional level of effector genes from secondary metabolism; Glombitza S et al.; Plant secondary metabolism significantly contributes to defensive measures against adverse abiotic and biotic cues . To investigate stress-induced, transcriptional alterations of underlying effector gene families, which encode enzymes acting consecutively in secondary metabolism and defense reactions, a DNA array (MetArray) harboring gene-specific probes was established . It comprised complete sets of genes encoding 109 secondary product glycosyltransferases and 63 glutathione-utilizing enzymes along with 62 cytochrome P450 monooxygenases and 26 ABC transporters . Their transcriptome was monitored in different organs of unstressed plants and in shoots in response to herbicides, UV-B radiation, endogenous stress hormones, and pathogen infection . A principal component analysis based on the transcription of these effector gene families defined distinct responses and crosstalk . Methyl jasmonate and ethylene treatments were separated from a group combining reactions towards two sulfonylurea herbicides, salicylate and an avirulent strain of Pseudomonas syringae pv . tomato . The responses to the herbicide bromoxynil and UV-B radiation were distinct from both groups . In addition, these analyses pinpointed individual effector genes indicating their role in these stress responses . A small group of genes was diagnostic in differentiating the response to two herbicide classes used . Interestingly, a subset of genes induced by P . syringae was not responsive to the applied stress hormones . Small groups of comprehensively induced effector genes indicate common defense strategies . Furthermore, homologous members within branches of these effector gene families displayed differential expression patterns either in both organs or during stress responses arguing for their non-redundant functions. J Bacteriol, 2005 Jan, 187(1), 329 - 35 Fate of predator and prey proteins during growth of Bdellovibrio bacteriovorus on Escherichia coli and Pseudomonas syringae prey; Barel G et al.; A two-dimensional electrophoretic analysis of protein distribution followed by identification of selected proteins by mass spectrometry was performed on fresh bdellovibrio cultures containing attack phase cells of the predatory bacterium Bdellovibrio bacteriovorus strain 109J-1 and the remains of an Escherichia coli or a Pseudomonas syringae pv . tomato prey . Cleavage of the peptidoglycan-associated outer membrane proteins (OMPs) OmpA in E . coli and OprF in P . syringae occurred in both prey . The tryptic peptides obtained from the cleavage products of OmpA and OprF were all located within the 19-kDa pronase-resistant N-terminal parts of the corresponding proteins . The predator cell fraction was separated from the prey ghosts in fresh bdellovibrio cultures by centrifugation on a Percoll-sucrose cushion . Proteins from each fraction were separated by two-dimensional electrophoresis and identified by mass spectrometric analysis . As no prey OMP could be detected in the predator cell fraction, it was concluded that prey OMPs are not transferred to the predator, as had been suggested previously . However, a protein from the predator was found bound to ghost cell envelopes . This protein may correspond to a protein earlier suggested to be associated with the prey outer or cytoplasmic membranes . Along with recently described polypeptides from B . bacteriovorus strains 100 and 114, it forms a new family of putative outer membrane proteins. Bone Marrow Transplant . 2004 Dec 13; {Epub ahead of print} A novel reduced-intensity stem cell transplant regimen for nonmalignant disorders; Shenoy S et al.; Summary:Bone marrow transplantation (BMT) benefits nonmalignant diseases but is limited by regimen-related toxicity, graft-versus-host disease (GVHD), donor availability, and graft rejection (GR) . To overcome some of these barriers, we developed a new conditioning strategy for these patients . In total, 16 patients received Campath-1H (33/48 mg; days -21 to -19), fludarabine (150 mg/m(2); days -8 to -4), melphalan (140/70 mg/m(2); day -3), and transplant using related/unrelated stem cells . GVHD prophylaxis included cyclosporine/methylprednisolone for cord cells . Other recipients also received methotrexate . Risk factors for GR included multiple transfusions (6), low stem cell numbers (1), and immunologic/metabolic disorders (3) . Donor engraftment was present in 14/16 recipients . Neutrophils (ANC>0.5 x 10(9)/l) and platelets (>50 x 10(9)/l) engrafted at a median of 13 and 24 days . Two patients died of Pseudomonas sepsis prior to engraftment, one of CMV disease, and another of intracranial hemorrhage . With median follow-up of 281 days (78-907), 12/16 are stable/improved, or cured . Acute GVHD was absent (n=10) or mild and transient (grade1-2 skin) (n=4) . There was no chronic GVHD . Toxicities were predominantly early infections within 100 days, and correlated with lymphopenia (CD4+ T and B cells) . Stable engraftment and low incidence of significant GVHD, irrespective of age or stem cell source, make this reduced-intensity regimen attractive for nonmalignant disorders.Bone Marrow Transplantation advance online publication, 13 December 2004; doi:10.1038/sj.bmt.1704795. J Microbiol Methods, 2005 Feb, 60(2), 235 - 45 Construction and comparison of Escherichia coli whole-cell biosensors capable of detecting aromatic compounds; Kim MN et al.; The XylR regulatory protein is a transcription factor involved in the BTEX (benzene, toluene, ethylbenzene, and xylene) degradation pathway in Pseudomonas species . When XylR-dependent stimulation of transcription from a plasmid containing XylR and its cognate promoters Pr and Pu was monitored as firefly luciferase activities in Escherichia coli, a notably high level of basal activity was observed in the absence of inducers . To improve the response specificity of XylR in this system, two related but different promoters were tested for their activities; the XylS activator promoter Ps and the DmpR activator promoter Po . Po with the deletion of its own upstream activating sequences (UASs; Po') showed a very low level of basal activity compared to Pu and Ps . The maximum level with the addition of inducers was increased 3151-fold by o-xylene with Po', while it was 31.5 and 74.1 fold by m-xylene with Pu and Ps, respectively . Gel mobility shift assay showed that the purified XylR without inducers can bind to Pr/Pu but not to Pr/Po', implying that XylR multimerization with Pr/Pu could be formed for initiation of transcription in this system . The data suggest that Po' can be an excellent alternative in constructing a signal-intensified, whole-cell biosensor in response to the xenobiotics. J Microbiol Methods, 2005 Feb, 60(2), 217 - 24 Quantification of the Pseudomonas population in New Zealand soils by fluorogenic PCR assay and culturing techniques; Lloyd-Jones G et al.; The genus Pseudomonas contains fast-growing nutritionally versatile bacteria that are able to utilize a wide variety of carbon sources . The ubiquity of the genus has been highlighted by conventional microbiology and the genus is well represented in collections of cultured bacteria . Here we evaluate the Pseudomonas population in New Zealand soils by comparing a culture-independent (real-time PCR combined with fluorescent TaqMan technology) with a culture-dependent (Gould's S1) population estimate . We show that cultivated fluorescent pseudomonads are not numerically dominant and represent a small proportion of <1% of the total Pseudomonas population, and that the total Pseudomonas population itself represents only a small proportion of <1% of the total bacterial population. Plant J, 2004 Dec, 40(6), 931 - 41 Role of chloroplast trienoic fatty acids in plant disease defense responses; Yaeno T et al.; Trienoic fatty acids (TAs) are the major polyunsaturated fatty acid species in the membrane lipids in plant cells . TAs are crucial for the adaptation to abiotic stresses, especially low- or high-temperature stress . We show that TAs in chloroplast membrane lipids are involved in defense responses against avirulent bacterial pathogens . Avirulent pathogen invasion of plants induces a transient production of reactive oxygen intermediates (ROI), programmed cell death and subsequent disease resistance . The Arabidopsis fad7fad8 mutation, which prevents the synthesis of TAs in chloroplast lipids, caused the reduction in ROI accumulation in leaves inoculated with Pseudomonas syringae pv . tomato DC3000 (avrRpm1) . Linolenic acid, the most abundant TA, activated the NADPH oxidase that is responsible for ROI generation . TAs were transferred from chloroplast lipids to extrachloroplast lipids coincident with ROI accumulation after inoculation with Pst DC3000 (avrRpm1) . Furthermore, the fad7fad8 mutant exhibited reduced cell death and was compromised in its resistance to several avirulent P . syringae strains . These results suggest that TAs derived from chloroplast lipids play an important role in the regulation of plant defense responses. Acta Crystallogr D Biol Crystallogr, 2004 Dec, 60(Pt 12 Pt 2), 2340 - 2 Epub 2004 Dec. Crystallization and preliminary crystallographic analysis of the 2'-aminobiphenyl-2,3-diol 1,2-dioxygenase from the carbazole-degrader Pseudomonas resinovorans strain CA10; Iwata K et al.; CarBaBb, the class III extradiol dioxygenase involved in carbazole degradation by Pseudomonas resinovorans CA10, was crystallized at 278 K by the hanging-drop vapour-diffusion method using PEG MME 550 as a precipitant . The crystals had a transparent thin square-pillar shape and belonged to space group P2(1)2(1)2, with unit-cell parameters a = 122.8, b = 144.6, c = 49.2 A, alpha = beta = gamma = 90 degrees . The crystals diffracted to a maximum resolution of 1.9 A and gave a data set with an overall R(merge) of 5.7% and a completeness of 98.6% . The V(M) value was 2.52 A(3) Da(-1), which indicated a solvent content of 51.2%. Anal Biochem, 2005 Jan 1, 336(1), 117 - 24 A colorimetric determination for glycosidic and bile salt-based detergents: applications in membrane protein research; Urbani A et al.; Detergents are crucial to the isolation of integral membrane proteins . During membrane protein purification, it is useful to accurately quantify detergent, especially if concentration steps have been used . Previously, this has been difficult and time-consuming . We present a simple, rapid, and sensitive procedure for the quantification of glycosidic and bile salt-based detergents such as dodecylmaltoside, octylglucoside, and CHAPS . The method directly quantifies sugar or cholate moieties via colorimetric reactions with phenol and sulfuric acid . A number of detergents have been screened, and the assay has been validated in the presence of commonly used reagents . In addition to determining the overall detergent concentration in solution, the procedure allows accurate quantification of specific binding of glycosidic or bile salt-based detergents to purified membrane proteins . Both the colorimetric method and the radiometric 14C method were used to determine detergent binding to two integral membrane proteins: the cytochrome cbb3 oxidase from Pseudomonas stutzeri and the turkey beta-adrenergic receptor . Both methods gave similar results . After separating monomeric glycosidic detergent from micellar solutions by ultrafiltration, we used the colorimetric method to determine the concentration of monomeric detergent present . We observed that values obtained are in close agreement with previously determined critical micelle concentrations. J Am Soc Nephrol, 2004 Dec, 15(12), 3207 - 14 Short bacterial DNA fragments: detection in dialysate and induction of cytokines; Schindler R et al.; A number of bacterial cytokine-inducing substances (CIS) such as lipopolysaccharides (LPS) and exotoxins have been detected in dialysate and may contribute to inflammation in hemodialysis patients . Short DNA fragments, oligodeoxynucleotides (ODN) of 6 to 20 nucleotides, are able to bind to Toll-like receptors and are stimulatory on immune cells . ODN induce natural killer cell activity and induce IFN-gamma, TNF-alpha, and IL-6 from mononuclear cells . The presence of ODN in dialysate samples and bacterial cultures was investigated . ODN were extracted from fluids by adsorption to reverse-phase columns . ODN were detected in 18 of 20 investigated dialysate samples, in eight of 10 reverse-osmosis water samples, and in all cultures from various bacterial strains . The presence of bacterial DNA in dialysate was confirmed by PCR specific for bacterial tRNA gene sequences . Saline for intravenous use contained 0.02 +/- 0.01 microg/ml DNA, dialysate samples contained 0.28 +/- 0.02 microg/ml, and Pseudomonas cultures contained 1.0 +/- 0.03 microg/ml DNA . ODN from bacterial cultures were only partially removed by ultrafiltration and were able to diffuse through regular high-flux dialyzer membranes . Synthetic cytosine-guanosine dinucleotide-containing ODN were able to induce IL-6 in human mononuclear cells . It is concluded that short bacterial-derived DNA fragments are present in clinically used fluids, e.g., dialysate . These fragments are of sufficient small size to pass through dialyzer membranes . Bacterial DNA fragments may be an overlooked factor contributing to inflammation in hemodialysis patients. Plant Biol (Stuttg), 2004 Nov, 6(6), 664 - 72 Inhibition of phosphoinositide-specific phospholipase C results in the induction of pathogenesis-related genes in soybean; Chou WM et al.; The inositol 1,4,5-trisphosphate (IP3) content is decreased in soybean cells following infection with Pseudomonas syringae pv . glycinea (Psg) . In this investigation, a differential display approach was applied to isolate soybean genes that are transcriptionally up-regulated by the inhibition of phosphoinositide-specific phospholipase C (PI-PLC) activity and to study if the transcription of those genes is altered following Psg infection . Four genes, transcriptionally activated following treatment with the PI-PLC-specific inhibitor U-73122, were cloned . Three of the four genes were induced following infection with Psg . The transcripts of a hydrolase homologue (GmHy) were induced in the incompatible but not compatible soybean-Psg interaction . The transcripts of a putative ascorbate oxidase gene (GmAO) were induced in both compatible and incompatible interactions . GmHy and GmAO may represent new classes of pathogenesis-related genes . In addition to these two novel genes, homologues of PR-10 and polygalacturonase inhibitor protein (GmPR10 and GmPGIP, respectively) were identified . These two genes have previously been reported as pathogenesis-related . Transcripts of GmPR-10, but not GmPGIP, were induced in both compatible and incompatible soybean-Psg interactions . Induction of these genes, except for GmPGIP, following inhibition of PI-PLC by either the U-73122 treatment or bacterial infection suggests that PI-PLC may negatively regulate the expression of defence genes. Transplant Proc, 2004 Oct, 36(8), 2299 - 301 Surveillance of perioperative infections after adult living donor liver transplantation; Matsuo K et al.; AIM: This study was conducted to clarify the management of perioperative infectious complications after adult living donor liver transplantation (LDLT) . PATIENTS AND METHODS: Fourteen adult LDLT patients were enrolled in this study . We examined the occurrence of infectious complications in these cases and the relationships of infectious complications to UNOS status and MELD score . Surveillance culture and immunoserologic analyses were performed . From the results of these analyses, we made a diagram of infection surveillance using a matrix of time and sampling site . Using the diagram, we chose sensitive antibiotics as soon as possible . RESULTS: The infection site and its pathogen were able to be detected in four (28.5%) patients, all of whom had MRSA infections, together with lung aspergillosis in one case, pseudomonas pneumonia in another, and both in another . Two patients died of lung aspergillosis . Bacteria detected in the airway tended to spread to other sites during the postoperative period . In all four patients in whom infectious diseases were detected, and in a fifth patient in whom the site of infection was not known, the UNOS status was 1 . The MELD score was calculated in eight patients, six of whom had high MELD scores (>20) . CONCLUSION: Most cases were manageable by choosing and changing antibiotics and antifungal drugs according to the results of surveillance cultures twice a week . However, aspergillosis had an extremely poor prognosis . Patients with a high MELD score or low UNOS status, or both, showed poor prognosis; and in them, multiple drug resistance bacteria caused severe perioperative infectious complications. Cell Mol Biol (Noisy-le-grand), 2004 Jul, 50(5), 585 - 90 Siderotyping of Antarctic fluorescent pseudomonas strains; Geoffroy VA et al.; Five fluorescent Pseudomonas strains isolated from Antarctica have been previously recognized as producing three structurally different pyoverdines . In the present work, siderotyping procedures have been used to classify these strains, together with 1282 isolates of different origins, into siderovars . The strain biodiversity encountered within each siderovar, as well as the potential taxonomic value of the siderovars, are described and discussed . It is concluded that a majority of antarctic strains are commonly distributed worldwide . One strain, however, presenting a particular pyoverdine structure found in a unique other isolate, was apparently much more specific to cold environment. Mol Plant Microbe Interact, 2004 Nov, 17(11), 1250 - 8 Transcriptional regulation of components of the type III secretion system and effectors in Pseudomonas syringae pv . phaseolicola; Thwaites R et al.; Quantitative real-time polymerase chain reaction was used with specific TaqMan probes to examine transcription of selected hrp and effector genes in Pseudomonas syringae pv . phaseolicola strains 1448A (race 6) and 1449B (race 7) . Transcripts examined were from genes encoding the regulators hrpR and hrpL, core structural components of the type III secretion system (TTSS) hrcC, hrcJ, hrcN, hrcU, and hrpA; the first open-reading frame of each hrp operon, including hrpF, hrpJ, hrpP, and hrpY, and also secreted effectors hrpZ, avrPphE, avrPphF, and virPphA . All genes were induced by incubation in a minimal medium and showed patterns of expression indicating regulation by HrpRS and HrpL . Basal mRNA levels and the timing of accumulation of transcripts after induction differed significantly, suggesting the operation of additional regulatory elements . However, no clear transcriptional hierarchy emerged to explain the ordered construction of the TTSS . Quantitative analysis confirmed that the rates and levels of transcript accumulation within the first 2 h after inoculation were considerably higher in planta than in vitro, and indicated that plant cell wall contact may enhance transcription of TTSS and effector genes in P . syringae pv . phaseolicola . The low-abundance hrcU mRNA had a half-life of 16.5 min, whereas other transcripts had half-lives between 3 and 8 min. J Pak Med Assoc, 2004 Oct, 54(10), 499 - 503 Allogeneic bone marrow transplantation in beta-thalassaemia--single centre study; Hashmi KU et al.; OBJECTIVE: To evaluate outcome of allogeneic BMT in beta-Thalassaemia at the Armed Forces Bone Marrow Transplant Centre, Rawalpindi, Pakistan from August 2001 to November 2003 . METHODS: Nineteen patients with beta-Thalassaemia underwent allogeneic BMT/PBSC transplantation from HLA identical sibling donors . Patients were classified in three groups according to Pesaro (Italy) risk classification . Class-I (n = 9) and Class-II (n = 7) patients received conditioning with busulphan/cyclophosphamide, whereas Class-III (n = 3) patient received conditioning with hydroxyurea, azathioprine, fludarabine, along with Bu14 / Cy 200 . Cyclosporine, prednisolone and methotrexate were given for GvHD prophylaxis . Stem cells dose infused was >4.0 x 10(8)/kg body weight of the patient . RESULTS: Engraftment was achieved in all Class-I patients, whereas in Class-II and Class-III , graft rejection was observed in one patient from each class . Median time to achieve absolute neutrophil recovery (> 0.5 x 10(9)/l) was 13 days, platelet count (> 20 x 10(9)/1) was 15 days and reticulocyte count (>0.5%) was 15 days . Acute GvHD was observed in 15 patients . One patient developed grade IV GvHD (liver and skin) and died within 30 days post BMT . Post transplant infectious complications were pseudomonas septicemia, disseminated fungal infection, CMV pneumonia and tuberculosis . Three patients died of these complications during post transplant period (31-90 days) . Median stay in hospital was 25 days . CONCLUSION: Allogeneic BMT is the only curative therapy for beta-Thalassaemia patients, however the success rate can be increased if the patients are selected carefully and transplanted at an early age. Plant J, 2004 Dec, 40(5), 790 - 8 The Pseudomonas syringae type III effector AvrRpt2 promotes virulence independently of RIN4, a predicted virulence target in Arabidopsis thaliana; Lim MT et al.; AvrRpt2, an effector protein from Pseudomonas syringae pv . tomato (Pst), behaves as an avirulence factor that activates resistance in Arabidopsis thaliana lines expressing the resistance gene RPS2 . AvrRpt2 can also enhance pathogen fitness by promoting the ability of the bacteria to grow and to cause disease on susceptible lines of A . thaliana that lack functional RPS2 . The activation of RPS2 is coupled to the AvrRpt2-induced disappearance of the A . thaliana RIN4 protein . However, the significance of this RIN4 elimination to AvrRpt2 virulence function is unresolved . To clarify our understanding of the contribution of RIN4 disappearance to AvrRpt2 virulence function, we generated new avrRpt2 alleles by random mutagenesis . We show that the ability of six novel AvrRpt2 mutants to induce RIN4 disappearance correlated well with their avirulence activities but not with their virulence activities . Moreover, the virulence activity of wild-type AvrRpt2 was detectable in an A . thaliana line lacking RIN4 . Collectively, these results indicate that the virulence activity of AvrRpt2 in A . thaliana is likely to rely on the modification of host susceptibility factors other than, or in addition to, RIN4. Z Naturforsch {C}, 2004 Sep-Oct, 59(9-10), 613 - 8 The pyoverdins of Pseudomonas syringae and Pseudomonas cichorii; Bultreys A et al.; The structure elucidation of the cyclic (lactonic) forms of the pyoverdins with a succinamide side chain originally produced by the closely related species Pseudomonas syringae and P . cichorii is reported . Mass spectrometry and nuclear magnetic resonance analyses as well as the determination of the configuration of the amino acids after degradation indicate that these two pyoverdins differ only by the replacement of the first in-chain serine by glycine . The pyoverdins of P . syringae and P . cichorii and the dihydropyoverdin of P . syringae can be used by both species as siderophores. Proc R Soc Lond B Biol Sci, 2004 Nov 7, 271(1554), 2275 - 82 Reduced fecundity is the cost of cheating in RNA virus phi6; Dennehy JJ et al.; Co-infection by multiple viruses affords opportunities for the evolution of cheating strategies to use intracellular resources . Cheating may be costly, however, when viruses infect cells alone . We previously allowed the RNA bacteriophage phi6 to evolve for 250 generations in replicated environments allowing co-infection of Pseudomonas phaseolicola bacteria . Derived genotypes showed great capacity to compete during co-infection, but suffered reduced performance in solo infections . Thus, the evolved viruses appear to be cheaters that sacrifice between-host fitness for within-host fitness . It is unknown, however, which stage of the lytic growth cycle is linked to the cost of cheating . Here, we examine the cost through burst assays, where lytic infection can be separated into three discrete phases (analogous to phage life history): dispersal stage, latent period (juvenile stage), and burst (adult stage) . We compared growth of a representative cheater and its ancestor in environments where the cost occurs . The cost of cheating was shown to be reduced fecundity, because cheaters feature a significantly smaller burst size (progeny produced per infected cell) when infecting on their own . Interestingly, latent period (average burst time) of the evolved virus was much longer than that of the ancestor, indicating the cost does not follow a life history trade-off between timing of reproduction and lifetime fecundity . Our data suggest that interference competition allows high fitness of derived cheaters in mixed infections, and we discuss preferential encapsidation as one possible mechanism. Immunol Res, 2004, 30(3), 339 - 49 Therapeutic targeting of IL-4- and IL-13-responsive cells in pulmonary fibrosis; Jakubzick C et al.; Severe forms of idiopathic interstitial pneumonia (IIP), such as usual interstitial pneumonia (UIP), can be impervious to modern steroid and immunosuppressive treatment regimens, thereby emphasizing the need for novel effective therapies . Understanding the cytokine networks that may affect immune and structural cell activation and, hence, the progression of these fatal fibrotic diseases, has been a focus in our research . In this regard, we have examined the role of interleukin (IL)-4 and IL-13 and their respective receptor subunits in this process . Examination of clinical surgical lung biopsies (SLBs) showed that IIP is characterized by the abnormal, heightened expression of the receptor subunits that bind IL-4 and IL-13 . Specifically, IL-4Ralpha and IL-13Ralpha2 (the high-affinity IL-13 receptor subunit) was present in greater abundance in SLBs and fibroblasts from IIP patients compared with normal patients, who exhibited no evidence of pulmonary fibrosis . These clinical findings prompted us to investigate whether the targeting of pulmonary cell types that were highly responsive to IL-4 and IL-13 was a viable therapeutic option in IIP . Using a chimeric protein comprised of human IL-13 and a truncated version of an exotoxin from Pseudomonas (abbreviated IL13-PE), we observed that IL13-PE selectively targeted human pulmonary fibroblasts grown from IIP SLBs, whereas it had a minimal effect on fibroblasts grown from biopsies from normal patients . In murine models characterized by abnormal airway or interstitial fibrotic responses, the intranasal administration of IL13-PE significantly attenuated the fibrotic response through the targeting of IL-4Ralpha- and IL-13Ralpha2-expressing pulmonary cells, including monocytes, macrophages, and pulmonary fibroblasts . Together, these data demonstrate that IL-4 and IL-13 are required for the initiation and maintenance of pulmonary fibrosis, and highlight the importance of further investigation of anti-fibrotic therapeutics that prevent the action of both cytokines during clinical pulmonary fibrosis. Curr Biol, 2004 Nov 9, 14(21), 1897 - 906 VPEgamma exhibits a caspase-like activity that contributes to defense against pathogens; Rojo E et al.; BACKGROUND: Caspases are a family of aspartate-specific cysteine proteases that play an essential role in initiating and executing programmed cell death (PCD) in metazoans . Caspase-like activities have been shown to be required for the initiation of PCD in plants, but the genes encoding those activities have not been identified . VPEgamma, a cysteine protease, is induced during senescence, a form of PCD in plants, and is localized in precursor protease vesicles and vacuoles, compartments associated with PCD processes in plants . RESULTS: We show that VPEgamma binds in vivo to a general caspase inhibitor and to caspase-1-specific inhibitors, which block the activity of VPEgamma . A cysteine protease inhibitor, cystatin, accumulates to 20-fold higher levels in vpegamma mutants . Homologs of cystatin are known to suppress hypersensitive cell death in plant and animal systems . We also report that infection with an avirulent strain of Pseudomonas syringae results in an increase of caspase-1 activity, and this increase is partially suppressed in vpegamma mutants . Plants overexpressing VPEgamma exhibit a greater amount of ion leakage during infection with P . syringae, suggesting that VPEgamma may regulate cell death progression during plant-pathogen interaction . VPEgamma expression is induced after infection with P . syringae, Botrytis cinerea, and turnip mosaic virus, and knockout of VPEgamma results in increased susceptibility to these pathogens . CONCLUSIONS: We conclude that VPEgamma is a caspase-like enzyme that has been recruited in plants to regulate vacuole-mediated cell dismantling during cell death, a process that has significant influence in the outcome of a diverse set of plant-pathogen interactions. J Clin Microbiol, 2004 Nov, 42(11), 5403 - 5 An unexpected experimental pitfall in the molecular diagnosis of bacterial endophthalmitis; Ugahary L et al.; General primer-mediated ribosomal DNA amplification during endophthalmitis may improve the quality of diagnostic microbiology . However, extreme care needs to be taken not to introduce contaminating bacterial DNA during surgery procedures . The use of decontaminating iodine solutions can lead to such contamination due to the presence of DNA from Pseudomonas-like organisms. Appl Environ Microbiol, 2004 Nov, 70(11), 6665 - 9 Pore size dependence on growth temperature is a common characteristic of the major outer membrane protein OprF in psychrotrophic and mesophilic Pseudomonas species; Jaouen T et al.; Pseudomonas species adapt well to hostile environments, which are often subjected to rapid variations . In these bacteria, the outer membrane plays an important role in the sensing of environmental conditions such as temperature . In previous studies, it has been shown that in the psychrotrophic strain P . fluorescens MF0, the major porin OprF changes its channel size according to the growth conditions and could affect outer membrane permeability . Studies of the channel-forming properties of OprFs from P . putida 01G3 and P . aeruginosa PAO1 in planar lipid bilayers generated similar results . The presence of a cysteine- or proline-rich cluster in the central linker region is not essential for channel size modulations . These findings suggest that OprF could adopt two alternative conformations in the outer membrane and that folding is thermoregulated . In contrast, no difference according to growth temperature was observed for structurally different outer membrane proteins, such as OprE3 from the Pseudomonas OprD family of specific porins . Our results are consistent with the fact that the decrease in channel size observed at low growth temperature is a particular feature of the OprF porin in various psychrotrophic and mesophilic Pseudomonas species isolated from diverse ecological niches . The ability to reduce outer membrane permeability at low growth temperature could provide these bacteria with adaptive advantages. Carbohydr Res, 2004 Nov 15, 339(16), 2657 - 65 Structure of minor oligosaccharides from the lipopolysaccharide fraction from Pseudomonas stutzeri OX1; Leone S et al.; A minor oligosaccharide fraction was isolated after complete de-acylation of the lipooligosaccharide extracted from Pseudomonas stutzeri OX1 . The full structure of this oligosaccharide was obtained by chemical degradation, NMR spectroscopy and MALDI-TOF MS spectrometry . These experiments showed the presence of two novel oligosaccharides (OS1 and OS2): {structure: see text} where R=(S)-Pyr(-->4,6) in OS1 and alpha-Rha-(1-->3) in OS2 . All sugars are D-pyranoses, except Rha, which is L-pyranose . Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, Pyr is pyruvic acid, P is phosphate. Plasmid, 2004 Nov, 52(3), 230 - 6 Organization of the horizontally transferred pheBA operon and its adjacent genes in the genomes of eight indigenous Pseudomonas strains; Peters M et al.; Horizontal transfer of genes encoding phenol degradation (pheBA) in the environment has been previously described . Complete or partial phe-operon was redetected in plasmids of several indigenous Pseudomonas strains isolated from the river water . The sequences of up- and downstream regions of the acquired phe-DNA in eight different plasmids were analyzed . In all cases, miniature insertional elements or putative transposase genes were found suggesting transposase dependent pheBA integration into plasmids . In three cases, an open reading frame encoding homologue to the transcription regulator protein (CatR) of the pheBA operon was determined. J Bacteriol, 2004 Nov, 186(22), 7807 - 10 Long-term effect of mutagenic DNA repair on accumulation of mutations in Pseudomonas syringae B86-17; Zhang S et al.; Forty replicate lineages of Pseudomonas syringae B86-17 cells expressing the rulAB mutagenic DNA repair (MDR) determinant or the rulB::Km MDR-deficient mutant GWS242 were passaged through single-cell bottlenecks (60 cycles), with a UV radiation (UVR) exposure given to half of the lineages at the beginning of each cycle . After every 10th bottleneck cycle, single-colony isolates from all 80 lineages were subjected to 39 phenotypic screens, with newly arising mutations detected in 60 and 0% of UVR-exposed or non-UVR-exposed B86-17 lineages, respectively, by the 60th cycle . Cellular fitness, measured as growth rate in a minimal medium, of UVR-exposed lineages of both B86-17 and GWS242 after 60 cycles was not significantly different from that of the ancestral strains . Although UVR exposure and MDR activity increased the occurrence of mutations in cells, a significant reduction in overall fitness was not observed. Plant Cell Physiol, 2004 Sep, 45(9), 1335 - 41 His-404 and His-405 are essential for enzyme catalytic activities of a bacterial indole-3-acetyl-L-aspartic acid hydrolase; Chou JC et al.; Bacterial indole-3-acetyl-l-aspartic acid (IAA-Asp) hydrolase has shown very high substrate specificity compared with similar IAA-amino acid hydrolase enzymes found in Arabidopsis thaliana . The IAA-Asp hydrolase also exhibits, relative to the Arabidopsis thaliana-derived enzymes, a very high Vmax (fast reaction rate) and a higher Km (lower substrate affinity) . These two characteristics indicate that there are fundamental differences in the catalytic activity between this bacterial enzyme and the Arabidopsis enzymes . By employing a computer simulation approach, a catalytic residue, His-385, from a non-sequence-related zinc-dependent exopeptidase of Pseudomonas was found to structurally match His-405 of IAA-Asp hydrolase . The His-405 residue is conserved in all related sequences of bacteria and Arabidopsis . Point mutation experiments of this His-405 to seven different amino acids resulted in complete elimination of enzyme activity . However, point mutation on the neighboring His-404 to eight other residues resulted in reduction, to various degrees, of enzyme activity . Amino acid substitutions for His-404 also showed that this residue influenced the minor activity of the IAA-Asp hydrolase for the substrates IAA-Gly, IAA-Ala, IAA-Ser, IAA-Glu and IAA-Asn . These results show the value and potential of structural modeling for predicting target residues for further study and for directing bioengineering of enzyme structure and function. Acta Crystallogr D Biol Crystallogr, 2004 Nov, 60(Pt 11), 2110 - 3 Epub 2004 Oct 20. Structure of the phenazine biosynthesis enzyme PhzG; Parsons JF et al.; PhzG is a flavin-dependent oxidase that is believed to play a role in phenazine antibiotic synthesis in various bacteria, including Pseudomonas . Phenazines are chorismic acid derivatives that provide the producing organisms, including the opportunistic pathogen P . aeruginosa, with a competitive growth advantage . Here, the crystal structures of PhzG from both P . aeruginosa and P . fluorescens solved in an unliganded state at 1.9 and 1.8 A resolution, respectively, are described . Although the specific reaction in phenazine biosynthesis catalyzed by PhzG is unknown, the structural data indicates that PhzG is closely related to pyridoxine-5'-phosphate oxidase, the Escherichia coli pdxH gene product, which catalyzes the final step in pyridoxal-5'-phosphate (PLP) biosynthesis . A previous proposal suggested that the physiological substrate of PhzG to be 2,3-dihydro-3-hydroxyanthranilic acid (DHHA), a phenazine precursor produced by the sequential actions of the PhzE and PhzD enzymes on chorismate, and that two DHHA molecules dimerized in another enzyme-catalyzed reaction to yield phenazine-1-carboxylate . However, it was not possible to demonstrate any in vitro activity upon incubation of PhzG and DHHA . Interestingly, analysis of the in vitro activities of PhzG in combination with PhzF suggests that PhzF acts on DHHA and that PhzG then reacts with a non-aromatic tricyclic phenazine precusor to catalyze an oxidation/aromatization reaction that yields phenazine-1-carboxylate . It is proposed that phzG arose by duplication of pdxH and that the subtle differences seen between the structures of PhzG and PdxH correlate with the loss of the ability of PhzG to catalyze PLP formation . Sequence alignments and superimpositions of the active sites of PhzG and PdxH reveal that the residues that form a positively charged pocket around the phosphate of PLP in the PdxH-PLP complex are not conserved in PhzG, consistent with the inability of phosphorylated compounds to serve as substrates for PhzG. Clin Cancer Res, 2004 Oct 15, 10(20), 7079 - 87 Abrogation of head and neck squamous cell carcinoma growth by epidermal growth factor receptor ligand fused to pseudomonas exotoxin transforming growth factor alpha-PE38; Thomas SM et al.; PURPOSE: This study was undertaken to determine whether low intratumoral doses of the epidermal growth factor receptor ligand-transforming growth factor alpha (TGF-alpha) fused to Pseudomonas exotoxin (TGF-alpha-PE38)-abrogated head and neck squamous cell carcinoma (HNSCC) tumor growth in vitro and in vivo . EXPERIMENTAL DESIGN: In vitro cytotoxicity assays were carried out to determine the sensitivity of HNSCC cells to TGF-alpha-PE38 . TGF-alpha-PE38-treated HNSCC cells were examined by immunoblotting for cleaved poly(ADP-ribose) polymerase to evaluate apoptosis . Nude mice bearing established HNSCC xenografts were treated with several doses of TGF-alpha-PE38 to evaluate the antitumor efficacy in vivo . Tumor sections were stained with terminal deoxynucleotidyl transferase-mediated nick end labeling for apoptosis . To determine the effect of oral administration of TGF-alpha-PE38, gavage injections of TGF-alpha-PE38 were administered, and the esophagus and surrounding soft tissue were then stained for apoptotic cells . RESULTS: HNSCC cell lines examined were sensitive to low doses of TGF-alpha-PE38 (EC(50) in the range of 1.6 to 10 ng/mL) . HNSCC cells treated with TGF-alpha-PE38 undergo apoptosis . Antitumor effects were observed using 0.1 and 0.03 microg of TGF-alpha-PE38 administered intratumorally . At these doses, the treatment was well tolerated . Tumors treated with the toxin had a higher number of apoptotic cells compared with the control tumors . No apoptotic cells were observed in the pharyngoesophageal tissues of the mice after gavage administration of the toxin suggesting that the toxin could be orally administered without toxicity . CONCLUSIONS: These results indicate that topical or intratumoral administration of low doses of TGF-alpha-PE38 may demonstrate antitumor effects in HNSCC without associated systemic toxicity. Plant J, 2004 Nov, 40(4), 558 - 74 The BOS loci of Arabidopsis are required for resistance to Botrytis cinerea infection; Veronese P et al.; Three Botrytis-susceptible mutants bos2, bos3, and bos4 which define independent and novel genetic loci required for Arabidopsis resistance to Botrytis cinerea were isolated . The bos2 mutant is susceptible to B . cinerea but retains wild-type levels of resistance to other pathogens tested, indicative of a defect in a response pathway more specific to B . cinerea . The bos3 and bos4 mutants also show increased susceptibility to Alternaria brassicicola, another necrotrophic pathogen, suggesting a broader role for these loci in resistance . bos4 shows the broadest range of effects on resistance, being more susceptible to avirulent strain of Pseudomonas syringae pv . tomato . Interestingly, bos3 is more resistant than wild-type plants to virulent strains of the biotrophic pathogen Peronospora parasitica and the bacterial pathogen P . syringae pv . tomato . The Pathogenesis Related gene 1 (PR-1), a molecular marker of the salicylic acid (SA)-dependent resistance pathway, shows a wild-type pattern of expression in bos2, while in bos3 this gene was expressed at elevated levels, both constitutively and in response to pathogen challenge . In bos4 plants, PR-1 expression was reduced compared with wild type in response to B . cinerea and SA . In bos3, the mutant most susceptible to B . cinerea and with the highest expression of PR-1, removal of SA resulted in reduced PR-1 expression but no change to the B . cinerea response . Expression of the plant defensin gene PDF1-2 was generally lower in bos mutants compared with wild-type plants, with a particularly strong reduction in bos3 . Production of the phytoalexin camalexin is another well-characterized plant defense response . The bos2 and bos4 mutants accumulate reduced levels of camalexin whereas bos3 accumulates significantly higher levels of camalexin than wild-type plants in response to B . cinerea . The BOS2, BOS3, and BOS4 loci may affect camalexin levels and responsiveness to ethylene and jasmonate . The three new mutants appear to mediate disease responses through mechanisms independent of the previously described BOS1 gene . Based on the differences in the phenotypes of the bos mutants, it appears that they affect different points in defense response pathways. Eye Contact Lens, 2004 Oct, 30(4), 252 - 3; discussion 263-4 Will higher-Dk materials give better corneal refractive therapy results and fewer complications? Sakamoto R, Sugimoto K. PURPOSE: To evaluate the current status of materials in use for corneal refractive therapies . METHODS: The most up-to-date data available on rigid gas-permeable (RGP) lenses, corneal swelling, and corneal infection are reviewed . RESULTS: Despite 125 Dk/t units or greater, studies have shown differences in overnight swelling between RGP and silicone hydrogel materials . There is a small but significant overnight swelling with RGP lenses in this class when compared with silicone hydrogels, yet studies of Pseudomonas adherence to human epithelium have shown that RGP-wearing corneas bind the least bacteria . CONCLUSIONS: Corneal refractive therapies are on the verge of international acceptance as a standard alternative for treating refraction . Continually using newer, more permeable lens materials is a necessity to ensure safety, efficacy, and, in turn, long-term acceptance. Mol Plant Microbe Interact, 2004 Oct, 17(10), 1162 - 71 Overexpression of NtERF5, a new member of the tobacco ethylene response transcription factor family enhances resistance to tobacco mosaic virus; Fischer U et al.; A new member of the tobacco (Nicotiana tabacum) AP2/ERF (ethylene response factor) transcription factor family, designated NtERF5, has been isolated by yeast one-hybrid screening . In vitro, recombinant NtERF5 protein weakly binds GCC box cis-elements, which mediate pathogen-regulated transcription of several PR (pathogenesis related) genes . NtERF5 transcription is transiently activated by wounding, by infection with the bacterial pathogen Pseudomonas syringae, as well as by inoculation with Tobacco mosaic virus (TMV) . In contrast, NtERF5 transcription is not enhanced after application of salicylic acid, jasmonic acid, or ethylene . Constitutive overexpression of NtERF5 (ERF5-Oex) under control of the 35S promoter results in no visible alterations in plant growth or enhanced resistance to Pseudomonas infection . Furthermore, no constitutive expression of PR genes has been observed . In contrast, ERF5-Oex plants show enhanced resistance to TMV with reference to reduced size of local hypersensitive-response lesions and impaired systemic spread of the virus . Since, in TMV-infected ERF5-Oex plants, the viral RNA accumulates only up to 10 to 30% of the wild-type level, we suggest that NtERF5-regulated gene expression is controlling resistance to viral propagation . Previous research has demonstrated that overexpression of ERF genes enhances resistance to bacterial and fungal pathogens . Here, we provide further evidence that resistance to viral infection can be engineered by overexpression of ERF transcription factors. Mol Plant Microbe Interact, 2004 Oct, 17(10), 1095 - 102 Impact of temperature on in planta expression of genes involved in synthesis of the Pseudomonas syringae phytotoxin coronatine; Weingart H et al.; Coronatine (COR) is a chlorosis-inducing phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae . Confocal laser scanning microscopy was used to investigate in vitro and in planta expression of COR genes by two model organisms, P . syringae pv . glycinea PG4180, a pathogen of soybean, and P syringae pv . tomato DC3000, a pathogen of tomato and crucifers . Previously, it was shown in vitro that the cma operon involved in COR synthesis in PG4180 is expressed in a temperature-dependent manner, with maximal rates at 18 degrees C and low activity at 28 degrees C . However, nothing was known about the influence of temperature on the expression of COR biosynthetic genes in planta . Therefore, transcriptional fusions of the PG4180 and DC3000 cma promoter regions to a promoterless egfp gene were constructed and expressed in both P . syringae strains . The fluorescence patterns in response to temperature during growth of a strain in vitro were consistent with its COR production and the cma transcript abundance as revealed by RNA dot blot hybridization . Quantification of fluorescence indicated that cma promoter activity was dependent on the genetic background of the host strain . Expression of cma::egfp in PG4180 was temperature-dependent in minimal medium as well as inside the plant tissue . In contrast, transcription of the cma operon was not significantly affected by temperature in DC3000 . However, cells of DC3000 harboring the cma::egfp fusions showed higher levels of fluorescence when recovered from infected host plants compared with cells grown in minimal medium . These results indicate that the signals for induction of COR biosynthesis differ significantly in PG4180 and DC3000. Biochemistry, 2004 Oct 26, 43(42), 13328 - 39 Structural analysis of Pseudomonas 1-aminocyclopropane-1-carboxylate deaminase complexes: insight into the mechanism of a unique pyridoxal-5'-phosphate dependent cyclopropane ring-opening reaction; Karthikeyan S et al.; 1-Aminocyclopropane-1-carboxylate (ACC) deaminase is a pyridoxal 5'-phosphate (PLP) dependent enzyme catalyzing the opening of the cyclopropane ring of ACC to give alpha-ketobutyric acid and ammonia as the products . This ring cleavage reaction is unusual because the substrate, ACC, contains no abstractable alpha-proton and the carboxyl group is retained in the product . How the reaction is initiated to generate an alpha-carbanionic intermediate, which is the common entry for most PLP-dependent reactions, is not obvious . To gain insight into this unusual ring-opening reaction, we have solved the crystal structures of ACC deaminase from Pseudomonas sp . ACP in complex with substrate ACC, an inhibitor, 1-aminocyclopropane-1-phosphonate (ACP), the product alpha-ketobutyrate, and two d-amino acids . Several notable observations of these structural studies include the following: (1) a typically elusive gem-diamine intermediate is trapped in the enzyme complex with ACC or ACP; (2) Tyr294 is in close proximity (3.0 A) to the pro-S methylene carbon of ACC in the gem-diamine complexes, implicating a direct role of this residue in the ring-opening reaction; (3) Tyr294 may also be responsible for the abstraction of the alpha-proton from d-amino acids, a prelude to the subsequent deamination reaction; (4) the steric hindrance precludes accessibility of active site functional groups to the l-amino acid substrates and may account for the stereospecificity of this enzyme toward d-amino acids . These structural data provide evidence favoring a mechanism in which the ring cleavage is induced by a nucleophilic attack at the pro-S beta-methylene carbon of ACC, with Tyr294 as the nucleophile . However, these observations are also consistent with an alternative mechanistic possibility in which the ring opening is acid-catalyzed and may be facilitated by charge relay through PLP, where Tyr294 functions as a general acid . The results of mutagenesis studies corroborated the assigned critical role for Tyr294 in the catalysis. Genetics . 2004 Oct 16; {Epub ahead of print} Genetic diversity, recombination, and cryptic clades in Pseudomonas viridiflava infecting natural populations of Arabidopsis thaliana; Goss EM et al.; Species level genetic diversity and recombination in bacterial pathogens of wild plant populations have been nearly unexplored . Pseudomonas viridiflava is a common natural bacterial pathogen of Arabidopsis thaliana, for which pathogen defense genes and mechanisms are becoming increasing well known . The genetic variation contained within a worldwide sample of P . viridiflava collected from wild populations of A . thaliana was investigated using five genomic sequence fragments totaling 2.3 kb . Two distinct and deeply diverged clades were found within the P . viridiflava sample, each genetically diverse with synonymous variation as high as 9.3% in one of these clades . Isolates representing the two clades were found in close proximity in multiple populations, but one clade was absent from all European population samples . Within clades, there is evidence of frequent recombination within and between each sequenced locus and little geographic differentiation . Isolates from both clades were also found on a small sample of other herbaceous species in Midwest populations, indicating a possibly broad host range for P . viridiflava . The high levels of genetic variation and recombination together with a lack of geographic differentiation in this bacterial pathogen distinguish it from other bacterial plant pathogens for which intraspecific variation has been examined. Mol Cell Biol, 2004 Nov, 24(21), 9487 - 97 Identification of the proteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation factor 2; Liu S et al.; Diphthamide, a posttranslational modification of translation elongation factor 2 that is conserved in all eukaryotes and archaebacteria and is the target of diphtheria toxin, is formed in yeast by the actions of five proteins, Dph1 to -5, and a still unidentified amidating enzyme . Dph2 and Dph5 were previously identified . Here, we report the identification of the remaining three yeast proteins (Dph1, -3, and -4) and show that all five Dph proteins have either functional (Dph1, -2, -3, and -5) or sequence (Dph4) homologs in mammals . We propose a unified nomenclature for these proteins (e.g., HsDph1 to -5 for the human proteins) and their genes based on the yeast nomenclature . We show that Dph1 and Dph2 are homologous in sequence but functionally independent . The human tumor suppressor gene OVCA1, previously identified as homologous to yeast DPH2, is shown to actually be HsDPH1 . We show that HsDPH3 is the previously described human diphtheria toxin and Pseudomonas exotoxin A sensitivity required gene 1 and that DPH4 encodes a CSL zinc finger-containing DnaJ-like protein . Other features of these genes are also discussed . The physiological function of diphthamide and the basis of its ubiquity remain a mystery, but evidence is presented that Dph1 to -3 function in vivo as a protein complex in multiple cellular processes. Org Biomol Chem, 2004 Oct 21, 2(20), 2942 - 50 Epub 2004 Sep 16. Synthetic 6-aryl-2-hydroxy-6-ketohexa-2,4-dienoic acid substrates for C-C hydrolase BphD: investigation of a general base catalytic mechanism; Speare DM et al.; A chemical synthesis of the 2-hydroxy-6-ketohexa-2,4-dienoic acid intermediates on bacterial meta-cleavage pathways has been established, using a Heck coupling strategy . Coupling of ethyl 3-bromo-2-acetoxyacrylate with 1-aryl vinyl ketals or 1-aryl allylic alcohols proceeded in 70-90% yield . Heck coupling with an alkyl vinyl ketal was also successful, allowing the synthesis of an alkyl-substituted ring fission intermediate . The synthetic ring fission intermediates were used to investigate the enzymatic reaction catalysed by C-C hydrolase BphD from Pseudomonas LB400 . A reduced substrate analogue 2,6-dihydroxy-6-phenylhexa-2,4-dienoic acid was processed enzymatically to benzaldehyde by C-C hydrolase BphD, consistent with a catalytic mechanism involving general base-catalysed attack of water to give a gem-diol intermediate, and not consistent with a nucleophilic mechanism . A series of para-substituted 2-hydroxy-6-keto-6-phenylhexa-2,4-dienoic acid substrates were assayed against BphD, and the derived Hammett plot (rho=-0.71) is consistent with a departing carbanion in the transition state for C-C cleavage. J Agric Food Chem, 2004 Oct 20, 52(21), 6552 - 6 Solvent extraction characterization of bioavailability of atrazine residues in soils; Barriuso E et al.; Characterization of pesticide bioavailability, particularly in aged soils, is of continued interest because this information is necessary for environmental risk assessment . The objective of this study was to correlate atrazine residue bioavailability in aged soils, as determined by solvent extraction methods, to atrazine mineralization by an atrazine-degrading bacterium . Webster clay loam and Zimmerman fine sand soils were treated with UL-ring-labeled {14C}atrazine and incubated for up to 8 weeks . At the end of each incubation period, soils were either not extracted, extracted with 0.01 M CaCl2, or extracted with 0.01 M CaCl2/aqueous methanol . Soils were then inoculated with the bacterium Pseudomonas sp . strain ADP, which is capable of rapidly mineralizing the atrazine ring . This allowed for the evaluation of the bioavailability of aged atrazine residues without the contribution of atrazine desorption from soil . Results of these studies indicated that the amounts of atrazine residues in aged soils extracted by 0.01 M CaCl2 and aqueous methanol were correlated to amounts of atrazine mineralized by Pseudomonas sp . strain ADP . Consequently, 0.01 M CaCl2/methanol extractable atrazine in aged soils may be used to estimate bioavailable residues, and this technique may be useful to determine the bioavailability of other compounds in soils, especially other triazine herbicides . FEMS Microbiol Lett, 2004 Oct 15, 239(2), 309 - 18 Heterologous expression of alkene monooxygenase components from Xanthobacter autotrophicus Py2 and reconstitution of the active complex; Champreda V et al.; The coupling protein and ferredoxin from Xanthobacter autotrophicus Py2 alkene monooxygenase (Xamo) have been functionally expressed in both N-terminal affinity tagged fusion and native forms in Escherichia coli . However, attempts to express the NADH-oxidoreductase and oxygenase, always resulted in the production of inactive, insoluble proteins . Nevertheless, the recombinant reductase from the toluene 4-monooxygenase of Pseudomonas mendocina KR1 was found to functionally complement the Xamo system . In vitro reconstitution, using the recombinant coupling protein and other components purified from the wild type, showed that steady-state epoxidation rate and coupling efficiency were dependent on the relative concentration of Xamo components in the reaction . The optimal molar stoichiometric ratio of Xamo components was determined to be approximately 1:0.25-0.3:2:2 (oxygenase hexamer:reductase:ferredoxin:coupling protein), suggesting the formation of an efficient catalytic complex at the minimal stoichiometric ratio to saturate the probable two-fold symmetry binding sites on the oxygenase. J Biochem Mol Biol, 2004 May 31, 37(3), 343 - 50 Molecular characterization of a thiJ-like gene in Chinese cabbage; Oh KJ et al.; A cDNA clone for a salicylic acid-induced gene in Chinese cabbage (Brassica rapa subsp . pekinensis) was isolated and characterized . The cabbage gene encoding a protein of 392 amino acids contained a tandem array of two thiJ-like sequences . ThiJ is a thiamin biosynthesis enzyme that catalyzes the phosphorylation of hydroxymethylpyrimidine (HMP) to HMP monophosphate . Although the cabbage gene shows a similarity to bacterial thiJ genes, it also shares a similarity with the human DJ-1, a multifunctional protein that is involved in transcription regulation, male fertility, and parkinsonism . The cabbage thiJ-like gene is strongly induced by salicylic acid and a nonhost pathogen, Pseudomonas syringae pv . tomato, which elicits a hypersensitive response in Chinese cabbage . Treatment of the cabbage leaves with BTH, methyl jasmonate, or ethephon showed that the cabbage thiJ-like gene expression is also strongly induced by BTH, but not by methyl jasmonate or ethylene . This indicates that the cabbage gene is activated via a salicylic acid-dependent signaling pathway . Examination of the tissue-specific expression revealed that the induction of the cabbage gene expression by BTH occurs in the leaf, stem, and floral tissues but not in the root. Mol Microbiol, 2004 Oct, 54(2), 353 - 65 HopPtoN is a Pseudomonas syringae Hrp (type III secretion system) cysteine protease effector that suppresses pathogen-induced necrosis associated with both compatible and incompatible plant interactions; Lopez-Solanilla E et al.; Pseudomonas syringae pv . tomato DC3000 causes bacterial speck disease in tomato, and it elicits the hypersensitive response (HR) in non-host plants such as Nicotiana tabacum and Nicotiana benthamiana . The compatible and incompatible interactions of DC3000 with tomato and Nicotiana spp., respectively, result in plant cell death, but the HR cell death occurs more rapidly and is associated with effective plant defense . Both interactions require the Hrp (HR and pathogenicity) type III secretion system (TTSS), which injects Hop (Hrp outer protein) effectors into plant cells . Here, we demonstrate that HopPtoN is translocated into tomato cells via the Hrp TTSS . A hopPtoN mutant produced eightfold more necrotic 'speck' lesions on tomato leaves than did DC3000, but the mutant and the wild-type strain grew to the same level in infected leaves . In non-host N . tabacum leaves, the hopPtoN mutant produced more cell death, whereas a DC3000 strain overexpressing HopPtoN produced less cell death and associated electrolyte leakage in comparison with wild-type DC3000 . Transient expression of HopPtoN via infection with a PVX viral vector enabled tomato and N . benthamiana plants to tolerate, with reduced disease lesions, challenge infections with DC3000 and P . syringae pv . tabaci 11528, respectively . HopPtoN showed cysteine protease activity in vitro, and hopPtoN mutants altered in the predicted cysteine protease catalytic triad (C172S, H283A and D299A) lost HR suppression activity . These observations reveal that HopPtoN is a TTSS effector that can suppress plant cell death events in both compatible and incompatible interactions. Plant J, 2004 Nov, 40(3), 366 - 75 Two Arabidopsis srfr (suppressor of rps4-RLD) mutants exhibit avrRps4-specific disease resistance independent of RPS4; Kwon SI et al.; RPS4 specifies the Arabidopsis disease resistance response to Pseudomonas syringae pv . tomato expressing avrRps4 and was cloned based on the identification of RLD as a naturally occurring susceptible accession . To dissect the molecular and genetic basis of disease resistance, we used a genetic approach to identify suppressor mutations that reactivate the avrRps4-triggered defense response in RLD . In this report, we describe two non-allelic srfr (suppressor of rps4-RLD) mutants, srfr1 and srfr3, that were susceptible to virulent P . syringae pv . tomato strain DC3000, but resistant to DC3000 expressing avrRps4 . In quantitative bacterial growth assays, growth of DC3000 was similar in wild-type control and both mutant lines, indicating that basal resistance was not enhanced in srfr1 and srfr3 . Growth of DC3000 (avrRps4) was approximately 30-fold lower in srfr1 and srfr3 than in RLD, but intermediate compared with fully resistant Col-0 and transgenic RLD containing RPS4-Col . The srfr1 and srfr3 mutants did not develop spontaneous lesions prior to inoculation or constitutively express the pathogenesis-related gene PR-1 . Therefore, srfr1 and srfr3 constitute novel avr-specific mutants that differ from previously described Arabidopsis mutants with elevated disease resistance . The srfr1 and srfr3 mutations were recessive, and both mapped to the bottom of chromosome IV . Genetic analysis indicated that resistance in srfr1 and srfr3 was independent of the rps4-RLD allele, but dependent on a second gene in RLD . We propose that SRFR1 and SRFR3 are negative regulators of avrRps4-triggered gene-for-gene disease resistance. Org Lett, 2004 Oct 14, 6(21), 3759 - 62 Regioselective enzymatic acylation of beta-L-2'-deoxynucleosides: application in resolution of beta-D/L-2'-deoxynucleosides; Garcia J et al.; {reaction: see text} A practical synthesis of beta-L-3'- and beta-L-5'-O-levulinyl-2'-deoxynucleosides has been described for the first time through enzymatic acylation and/or hydrolysis processes . It is noteworthy that the different behavior exhibited by Pseudomonas cepacia lipase in the acylation of D- and L-nucleosides allows the parallel kinetic resolution of D/L-nucleosides. Bioorg Khim, 2004 Jul-Aug, 30(4), 394 - 9 {Tryptophan 7-halogenase from Pseudomonas aureofaciens ACN strain: gene cloning and sequencing and the enzyme expression}; Micro construction of poly(epsilon-caprolactone)/poly(L-lactic acid) blend film by solution casting under microwave irradiation; Department of Polymer Science, College of Chemistry and Molecular Science, Wuhan University, Key Laboratory of Biomedical Polymers, Ministry of Education, Wuhan 430072, ChinaThe micro construction of poly(epsilon-caprolactone) (PCL) and poly(L-lactic acid) (PLLA) blend films fabricated by solution casting under microwave irradiation was investigated by selective enzymatic degradation and scanning electron microscopy (SEM).The results were totally different from the blends obtained by conventional methods . The blend was more homogeneous and the PCL continuous phase more compact as no spherulites and tiny zone separation were observed from the film surface and no PCL network was observed inside the film, and the degradation of a PCL plank by Pseudomonas lipase was significantly retarded . The distributed PLLA micro spheres were enlarged and amorphous . The thermal behavior of the blend by microwave heating revealed that PCL and PLLA underwent a melting process, which induced the variations of the PCL phase and PLLA spheres.The weight loss caused by degradation of the PCL/PLLA blend obtained by conventional methods (B50c) is greater than that of the blend obtained by microwave methods (B50m), which reflects the change in morphology from a loose PCL network (B50c) to a dense PCL plank (B50m). Macromol Biosci, 2004 Mar 15, 4(3), 296 - 307 Crystal structure, thermal behavior and enzymatic degradation of poly(tetramethylene adipate) solution-grown chain-folded lamellar crystals; Iwata T et al.; Solution-grown chain-folded lamellar single crystals of poly(tetramethylene adipate) (PTMA) were prepared from a dilute solution of 2-methyl-1-propanol by isothermal crystallization . PTMA crystals were hexagonal-shaped and polyethylene decoration of the crystals resulted in a "six cross-sector" surface morphology and showed that the average direction of chain folding is parallel to the crystal growth planes of {110} and {010} . Chain-folded lamellar crystals gave well-resolved electron diffraction diagrams corresponding to all the equatorial reflections of the X-ray fiber diagram obtained from stretched PTMA melt-quenched film (beta structure) . The unit cell parameters of the beta structure of PTMA were determined as a = 0.503 nm, b = 0.732 nm and c (fiber axis) = 1.442 nm with an orthorhombic crystal system . The fiber repeat distance is appropriate for an all-trans backbone conformation for the straight stems . The setting angle, with respect to the a axis, is +/-46 degrees for the corner and center chains . Thermal behavior of lamellar crystals has been investigated by means of transmission electron microscopy (TEM) and atomic force microscopy (AFM) . The lamellar thickness at the edges of the crystal increased after thermal treatment with taking the molecular chains into recrystallization parts; the holes then opened up at the thickening front of the crystal . The morphological changes of lamellar crystals after enzymatic degradation by Lipase type XIII from Pseudomonas sp . and water-soluble products were characterized by TEM, AFM, gel permeation chromatography, high performance liquid chromatography and fast atom bombardment mass spectrometry . The degradation progressed mainly from the edges of the lamellar crystals without decreasing the molecular weights and the lamellar thicknesses . The central portion of single crystals was often degraded by enzymatic attacks . This result combined with thermal behavior indicates that the loosely chain-packing region exists inside the single crystal, and that molecular chains in this region have higher mobility against thermal and enzymatic treatments. J Biomed Biotechnol, 2004, 2004(4), 219 - 226 Production of Prednisolone by Pseudomonas oleovorans Cells Incorporated Into PVP/PEO Radiation Crosslinked Hydrogels; Abd El-Hady A et al.; In order to rise the yield of prednisolone from hydrocortisone, the Pseudomonas oleovorans cells were entrapped into radiation crosslinked poly (vinyl pyrrolidone)/poly(ethylene oxide) (PVP/PEO) hydrogel of different gel contents . The factors affecting the gel content and swelling behavior of the polymeric gel, such as polymer composition, polymer blend concentration, and irradiation doses, were investigated . The formation of gels having a good strength with the ability to retain a desirable amount of water in their three-dimensional network can be achieved by using PVP/PEO copolymer of composition $(90:10)$ and concentration of 15% prepared at 20 kGy irradiation dose . At these conditions the prepared hydrogel is considered the most favorable one that gave the highest hydrocortisone bioconversion and prednisolone yield, 81% and 62.8%, respectively . The improvement of prednisolone yield was also achieved by increasing substrate concentration . Maximum hydrocortisone bioconversion (86.44) was obtained at 18 hours by using substrate concentration of 30 mg . Reusability of immobilized Pseudomonas oleovorans entrapped into PVP/PEO copolymer hydrogel was studied . The results indicated that the transformation capacity of hydrocortisone to prednisolone highly increased by the repeated use of copolymer for 4 times . This was accompanied by an increase in prednisolone yield to 89% and the bioconversion of hydrocortisone was 98.8%. Appl Environ Microbiol, 2004 Oct, 70(10), 6147 - 56 Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities; Cardinale M et al.; ITSF and ITSReub, constituting a new primer set designed for the amplification of the 16S-23S rRNA intergenic transcribed spacers, have been compared with primer sets consisting of 1406F and 23Sr (M . M . Fisher and E . W . Triplett, Appl . Environ . Microbiol . 65:4630-4636, 1999) and S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 (L . Ranjard et al., Appl . Environ . Microbiol . 67:4479-4487, 2001), previously proposed for automated ribosomal intergenic spacer analysis (ARISA) of complex bacterial communities . An agricultural soil and a polluted soil, maize silage, goat milk, a small marble sample from the facade of the Certosa of Pavia (Pavia, Italy), and brine from a deep hypersaline anoxic basin in the Mediterranean Sea were analyzed with the three primer sets . The number of peaks in the ARISA profiles, the range of peak size (width of the profile), and the reproducibility of results were used as indices to evaluate the efficiency of the three primer sets . The overall data showed that ITSF and ITSReub generated the most informative (in term of peak number) and reproducible profiles and yielded a wider range of spacer sizes (134 to 1,387) than the other primer sets, which were limited in detecting long fragments . The minimum amount of DNA template and sensitivity in detection of minor DNA populations were evaluated with artificial mixtures of defined bacterial species . ITSF and ITSReub amplified all the bacteria at DNA template concentrations from 280 to 0.14 ng microl(-1), while the other primer sets failed to detect the spacers of one or more bacterial strains . Although the primer set consisting of ITSF and ITSReub and that of S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 showed similar sensitivities for the DNA of Allorhizobium undicula mixed with the DNA of other species, the S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 primer set failed to detect the DNA of Pseudomonas stutzeri. Mikrobiol Z, 2004 May-Jun, 66(3), 5 - 13 {Taxonomic analysis of Pseudomonas strains with uncertain taxonomic status}; Solution structure of T4moC et al.; IKB, Agricultural University of Norway, Box 5040, 1432 NLH, NorwayToluene 4-monooxygenase, a four-protein complex from Pseudomonas mendocina KR1, catalyzes the NADH- and O(2)-dependent hydroxylation of toluene to form p-cresol . The solution structure of the 112-amino-acid Rieske ferredoxin component, T4moC, was determined from 2D and 3D (1)H, (13)C, and (15)N NMR data . The structural model was refined through simulated annealing by molecular dynamics in torsion angle space with input from 1650 experimental restraints, including 1264 inter-proton distance restraints obtained from NOEs, 247 non-redundant intra-residue NOEs, 26 hydrogen bond restraints, and 113 dihedral angle ( phi, psi) restraints . The 20 calculated conformers that best satisfied the input restraints were submitted to refinement in explicit solvent to improve the stereochemical quality . With exclusion of ill-defined N- and C-terminal segments (Ser2; His111-Ser112) and residues near to the {2Fe-2S} cluster, the atomic root mean square deviation for the 20 conformers with respect to the mean coordinates was 1.09 A for the backbone and 1.60 A for all non-hydrogen atoms . The T4moC structure consists of 10 beta-strands arranged in the three anti-parallel beta-sheet topology observed in all Rieske {2Fe-2S} domain proteins . The S(gamma) of Cys45 and Cys64 and the N(delta1) of His47 and His67 provide the ligands to the {2Fe-2S} cluster of T4moC . (1)H-(15)N HSQC measurements show that both His47-N(epsilon2) and His67-N(epsilon2) are protonated at the pH of the NMR experiments . Comparisons are made between the present NMR structure, previous paramagnetic NMR studies of T4moC, and the X-ray structures of other members of the Rieske protein family. Clin Cancer Res, 2004 Sep 15, 10(18 Pt 1), 6231 - 8 Analysis of antitumor activity of an interleukin-13 (IL-13) receptor-targeted cytotoxin composed of IL-13 antagonist and Pseudomonas exotoxin; Kioi M et al.; We have shown previously that a chimeric fusion protein composed of human interleukin-13 (IL-13) and Pseudomonas exotoxin (PE), termed IL-13 cytotoxin (IL13-PE38), is specifically cytotoxic to various cancer cell lines and primary cell cultures derived from a variety of solid cancers . In addition, we have shown that IL-13 mutant IL-13E13K, in which glutamic acid (E) residue at position 13 of IL-13 molecule was substituted by a lysine (K), is a powerful antagonist of IL-13 and binds to IL-13 receptor with a higher affinity compared with wild-type IL-13 . In this study, we have generated an IL-13 cytotoxin IL13E13K-PE38, in which IL-13 antagonist is fused to PE to determine whether this molecule has improved cytotoxicity to tumor cells compared with wild type (wt)IL13-PE38 . Highly purified IL13E13K-PE38 was tested in various tumor cell lines including seven glioblastoma multiforme cell lines to compare its binding to the cells, in vitro cytotoxicity, in vivo antitumor activity, and safety in mouse model with wtIL13-PE38 . IL13E13K-PE38 bound to U251MG and IL-13Ralpha2 chain-transfected tumor cell lines with 3 to 10 times higher affinity compared with wtIL13-PE38 . However, IL13E13K-PE38 did not show higher cytotoxicity compared with wtIL13-PE38 in glioblastoma multiforme or any other cell lines tested . The antitumor activity of IL13E13K-PE38, when administered intraperitoneally to nude mice bearing U251 tumors, was also similar to wtIL13-PE38 . Some improvement in antitumor activity was observed when lower doses of IL13E13K-PE38 were injected intratumorally in subcutaneous tumors . These results indicate that in general, IL13E13K-PE38 mediates similar cytotoxicity and antitumor activity to wtIL13-PE38 despite its improved binding affinity to IL-13 receptors. Plant J, 2004 Oct, 40(2), 225 - 37 Overexpression of the plasma membrane-localized NDR1 protein results in enhanced bacterial disease resistance in Arabidopsis thaliana; Coppinger P et al.; Previous studies have established that mutations in the NDR1 gene in Arabidopsis thaliana suppress the resistance response of three resistance proteins, RPS2, RPM1, and RPS5, to Pseudomonas syringae pv . tomato (Pst) strain DC3000 containing the cognate effector genes, avrRpt2, avrRpm1, and avrpPhB, respectively . NDR1 is a plasma membrane (PM)-localized protein, and undergoes several post-translational modifications including carboxy-terminal processing and N-linked glycosylation . Expression of NDR1 under the NDR1 native promoter complements the ndr1-1 mutation, while overexpression of NDR1 results in enhanced resistance to virulent Pst . Sequence analysis and mass spectrometry suggest that NDR1 is localized to the PM via a C-terminal glycosylphosphatidyl-inositol (GPI) anchor . GPI modification would potentially place NDR1 on the outer surface of the PM, perhaps allowing NDR1 to act as a transducer of pathogen signals and/or interact directly with the pathogen. Plant J, 2004 Oct, 40(2), 213 - 24 Expression of RPS4 in tobacco induces an AvrRps4-independent HR that requires EDS1, SGT1 and HSP90; Zhang Y et al.; The Arabidopsis RPS4 gene belongs to the Toll/interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NB-LRR) class of plant resistance (R) genes . It confers resistance to Pseudomonas syringae carrying the avirulence gene avrRps4 . Transient expression of genomic RPS4 driven by the 35S promoter in tobacco leaves induces an AvrRps4-independent hypersensitive response (HR) . The same phenotype is seen after expression of a full-length RPS4 cDNA . This indicates that alternative splicing of RPS4 is not involved in this HR . The extent of HR is correlated with RPS4 protein levels . Deletion analyses of RPS4 domains show the TIR domain is required for the HR phenotype . Mutations in the P-loop motif of the NB domain abolish the HR . Using virus-induced gene silencing, we found that the cell death resulting from RPS4 expression is dependent on the three plant signalling components EDS1, SGT1 and HSP90 . All these data suggest that heterologous expression of an R gene can result in activation of cell death even in the absence of its cognate avirulence product, and provides a system for studying the RPS4 domains required for HR. Acta Crystallogr D Biol Crystallogr, 2004 Oct, 60(Pt 10), 1922 - 4 Epub 2004 Sep 23. Overexpression, purification and crystallization of bacteriocin LlpA from Pseudomonas sp . BW11M1; Parret AH et al.; LlpA is a bacteriocin produced by Pseudomonas sp . BW11M1 that shows remarkable similarity to a family of mannose-binding plant lectins . A His-tagged version of LlpA was recombinantly produced in Escherichia coli and purified to homogeneity . Single crystals were grown by vapour diffusion and belong to space group P2(1)2(1)2, with unit-cell parameters a = 150.5, b = 154.5, c = 34.2 A . The crystals diffract to at least 2.2 A using synchrotron radiation. J Mol Recognit, 2004 Nov-Dec, 17(6), 540 - 57 Synthetic peptide vaccine development: measurement of polyclonal antibody affinity and cross-reactivity using a new peptide capture and release system for surface plasmon resonance spectroscopy; Cachia PJ et al.; A method has been developed for measurement of antibody affinity and cross-reactivity by surface plasmon resonance spectroscopy using the EK-coil heterodimeric coiled-coil peptide capture system . This system allows for reversible capture of synthetic peptide ligands on a biosensor chip surface,