Jpn J Antibiot, 1997 Nov, 50(11), 862 - 70 {Efficacy and safety of sulbactam/cefoperazone for hepato-biliary infections}; Shinagawa N et al.; We studied efficacy and safety of sulbactam/cefoperazone (SBT/CPT) in the treatment of biliary tract infections in hospitalized patients at 26 hospitals from February 1993 to March 1995 . Secondary to dropout, 273 out of 338 patients entered in the study were evaluated, 127 patients with cholecystitis, 132 patients with cholangitis, and 14 patients with liver abscesses . Of these, 93 patients (34.1% had malignancy as an underlying disease . SBT/CPZ had an efficacy of 79.9% (218 patients; excellent: 52, good: 166), with the efficacy in patients with cholecystitis, cholangitis and liver abscess at 89.0% (113 patients), 77.3% (102 patients and 21.4% (3 patients), respectively . A significant difference (p < 0.05) was observed in the efficacy rates of patients with (59 patients {63.4%}) and without malignancy (159 patients {88.3%}) . A total of 84 strains were isolated from bile specimens of 53 patients, and the major isolates were Escherichia coli, Pseudomonas aeruginosa and Enterococcus spp . Two or more bacterial strains were isolated simultaneously in 20 patients . Mild or moderate side effect (allergic reaction including rash etc.) were noted in 4 patients (1.18%), and laboratory abnormalities (increased GOT, etc.) were in 16 patients (4.71%) out of the total 338 patients . This study clearly demonstrated that SBT/CPZ retains its excellent clinical efficacy and safety profile, throughout its use over the past decade. Cancer Res, 1998 Jun 15, 58(12), 2661 - 6 Systemic treatment with a recombinant erbB-2 receptor-specific tumor toxin efficiently reduces pulmonary metastases in mice injected with genetically modified carcinoma cells; Maurer-Gebhard M et al.; Receptor-mediated targeted tumor therapy is an important applied consequence of the studies on the genetic causes of cancer . These therapy concepts have to be evaluated in novel animal models that reflect the molecular aberrations found in human tumors . Here we introduce an animal model that allows the evaluation of drugs directed against a surface receptor that is frequently altered in primary human adenocarcinomas . Tumor toxins are polypeptides in which a tumor cell-specific recognition domain and a toxic effector domain have been joined by DNA recombination in vitro . Tumor cell recognition is contributed by a single-chain antibody domain specific for the extracellular domain of the erbB-2 receptor {scFv(FRP5)} and cytotoxicity by the enzymatically active domain of a bacterial exotoxin (exotoxin A from Pseudomonas aeruginosa) . The erbB-2 receptor is overexpressed in many primary human cancer cells and is a favorable target for directed tumor therapy . The fusion protein scFv(FRP5)-exotoxin A has previously been shown to be able to efficiently and specifically kill erbB-2 receptor-expressing tumor cells . We have investigated the potential of this tumor toxin to detect and eliminate metastasizing tumor cells upon systemic administration . Murine renal carcinoma cells genetically modified with human erbB-2 receptor and bacterial beta-galactosidase genes form large pulmonary metastases when injected into the tail vein of BALB/c mice . Administration of the tumor toxin over a 10-day time period starting 1 day after tumor cell transplantation totally suppressed the formation of metastases . The treatment of animals 11 days after tumor cell transplantation, allowing the establishment of many pulmonary metastases, led to a drastic reduction in their number and size. Eur Respir J, 1998 May, 11(5), 1053 - 9 Orientation and presence of epithelium are key to endotoxin-induced neutrophil migration; Peralta FM et al.; The mechanisms by which endotoxins mediate neutrophil transepithelial migration and lung inflammation are unclear . It was hypothesized that both the presence and orientation of epithelial cells are critical to endotoxin-induced neutrophil migration . Neutrophil migration was compared through naked filters and filters with A549 lung epithelial monolayers grown on the upper and lower surface of permeable filters to simulate the apical and basal directional movement of neutrophils, respectively . The endotoxin, Pseudomonas aeruginosa lipopolysaccharide, was placed below the filter, acting as either a basal or an apical stimulus . Endotoxin without serum failed to stimulate neutrophil migration . In the presence of 1% human serum, endotoxin-induced neutrophil migration through naked filters was dose dependent . Endotoxin-induced neutrophil migration across A549 monolayers was minimal when the monolayers were cultured on the upper surface of the filters (basal stimulus) . In contrast, neutrophil transepithelial migration was much greater and dependent on both dose and time when the monolayer was cultured on the lower surface of the filter (basal to apical neutrophil directional movement) . Furthermore, enhanced neutrophil transepithelial migration was greater with an apical than with a basal stimulus . Endotoxin-induced neutrophil transepithelial migration was markedly inhibited (>95%) by actinomycin D pretreatment of the monolayers, suggesting the necessity for intact protein synthesis capacity of the A549 cells . Thus, both the presence and orientation of airway epithelium are key in supporting endotoxin-mediated lung neutrophilic responses. J Microw Power Electromagn Energy, 1998, 33(2), 77 - 87 Synergistic effect of microwave heating and hydrogen peroxide on inactivation of microorganisms; Kuchma T; Escherichia coli K-12 isogenous strains and Pseudomonas aeruginosa 102 were used to study the synergistic effects of combined microwave heating at short-time processing with low concentrations of hydrogen peroxide . The effect of microwave heating to temperatures of 40, 50 and 60 degrees C, as well as the concentration of hydrogen peroxide (0.05, 0.08 and 0.1%), the sequence of the agents' use, the nature of microorganisms on the survival of cells, DNA damages and interaction factors were studied . A method of anomalous viscosity time dependencies (AVTD) was used for measurement of the changes of genome conformational state (GCS) simultaneously with bacterial survival determination . The synergistic effect of microwave heating and low concentrations of hydrogen peroxide was observed under combined application, and reached a maximum when the cells were exposed to microwave heating to 50 degrees C and 0.08% hydrogen peroxide simultaneously . Both maxima of cell destruction and DNA injuries have been achieved by successive exposure to (MW + 10 min H2O2) to 60 degrees C and 0.08% hydrogen peroxide . The mechanisms of synergistic effects, the role of a disturbance of DNA repair and the interaction of sublethal injuries caused by different agents are discussed. Appl Environ Microbiol, 1998 Jul, 64(7), 2686 - 90 Specific detection of Legionella pneumophila: construction of a new 16S rRNA-targeted oligonucleotide probe; Grimm D et al.; Based on comparative sequence analysis, we have designed an oligonucleotide probe complementary to a region of 16S rRNA of Legionella pneumophila which allows the differentiation of L . pneumophila from other Legionella species without cultivation . The specificity of the new probe, LEGPNE1, was tested by in situ hybridization to a total of four serogroups of six strains of L . pneumophila, five different Legionella spp . and three nonlegionella species as reference strains . Furthermore, L . pneumophila cells could be easily distinguished from Legionella micdadei and Pseudomonas aeruginosa cells by using in situ hybridization with probes LEGPNE1, LEG705, and EUB338 after infection of the protozoan Acanthamoeba castellanii. Mol Gen Genet, 1998 May, 258(3), 250 - 9 Characterization of ptxS, a Pseudomonas aeruginosa gene which interferes with the effect of the exotoxin A positive regulatory gene, ptxR; Colmer JA et al.; The complicated process of exotoxin A production by Pseudomonas aeruginosa is controlled by several genes . We have recently described a toxA positive regulatory gene, ptxR . We also proposed the presence of another gene which is adjacent to ptxR and interferes with ptxR function on exotoxin A production . In the presence of a fragment that carries the putative gene, the enhancement in exotoxin A production by ptxR was reduced threefold . In this study, we describe the characterization of this gene . Nucleotide sequence analysis of the 2.1-kbp fragment at the 5' end of ptxR revealed the presence of an open reading frame designated ptxS (the gene next to ptxR) which encodes a 37.4-kDa protein . The gene ptxS is transcribed in the opposite orientation to ptxR from the other DNA strand . The deduced amino acid sequence of ptxS exhibited a strong homology to several proteins of the GalR-LacI family of repressors . A putative helix-turn-helix DNA binding motif was identified at the amino-terminus region of PtxS . When PtxS was overexpressed in Escherichia coli using the T7 expression system, a single protein of 38-kDa molecular weight was detected . An isogenic mutant defective in ptxS was constructed in PAO1 using the gene replacement technique . The loss of ptxS resulted in a twofold increase in exotoxin A production compared to PAO1 . The effect of ptxS on ptxR was examined using a ptxR-lacZ fusion . In the presence of ptxS, the level of beta-galactosidase activity produced by the ptxR-lacZ fusion was significantly reduced . These results suggest that ptxS encodes a protein which negatively regulates ptxR expression in P . aeruginosa. J Biol Chem, 1998 Jul 3, 273(27), 16792 - 7 Protease IV, a unique extracellular protease and virulence factor from Pseudomonas aeruginosa; Engel LS et al.; Comparisons of virulence between a Pseudomonas parent strain and an isogenic mutant devoid of protease IV have demonstrated a significant role for this enzyme during infection . We have characterized purified Pseudomonas aeruginosa protease IV in terms of its biochemical and enzymatic properties, and found it to be a unique extracellular protease . The N-terminal decapeptide sequence of protease IV is not homologous with any published protein sequence . Protease IV has a molecular mass of 26 kDa, an isoelectric point of 8.70, and optimum enzymatic activity at pH 10.0 and 45 degreesC . Purified protease IV demonstrates activity for the carboxyl side of lysine-containing peptides and can digest a number of biologically important proteins, including immunoglobulin, complement components, fibrinogen, and plasminogen . Protease IV is not inhibited by thiol-, carboxyl-, or metalloproteinase inhibitors . The total loss of enzyme activity in the presence of N-p-tosyl-L-chloromethyl ketone and the partial inhibition of enzyme activity by diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride imply that protease IV is a serine protease . Inhibition by dithiothreitol and beta-mercaptoethanol suggests that intramolecular disulfide bonds are essential for enzyme activity . The characteristics of this enzyme suggest that inhibitors of serine proteases could be developed into a medication designed to arrest tissue damage during Pseudomonas infection. J Bacteriol, 1998 Jul, 180(13), 3467 - 9 Secretion of elastinolytic enzymes and their propeptides by Pseudomonas aeruginosa; Braun P et al.; Elastase of Pseudomonas aeruginosa is synthesized as a preproenzyme . The signal sequence is cleaved off during transport across the inner membrane and, in the periplasm, proelastase is further processed . We demonstrate that the propeptide and the mature elastase are both secreted but that the propeptide is degraded extracellularly . In addition, reduction of the extracellular proteolytic activity led to the accumulation of unprocessed forms of LasA and LasD in the extracellular medium, which shows that these enzymes are secreted in association with their propeptides . Furthermore, a hitherto undefined protein with homology to a Streptomyces griseus aminopeptidase accumulated under these conditions. Infect Immun, 1998 Jul, 66(7), 3443 - 6 Susceptibility of epithelial cells to Pseudomonas aeruginosa invasion and cytotoxicity is upregulated by hepatocyte growth factor; Fleiszig SM et al.; Normal cell polarity protects epithelial cells against Pseudomonas aeruginosa invasion and cytotoxicity . Using epithelial cell clones with selective defects in sorting of membrane constituents, and using hepatocyte growth factor pretreatment, we found that polarized susceptibility to P . aeruginosa can be altered without disrupting tight junctions . The results also showed that cellular susceptibility factors for invasion and cytotoxicity are not the same, although both are localized to the basolateral cell surface in polarized epithelial cells. Infect Immun, 1998 Jul, 66(7), 3242 - 9 In vitro cellular toxicity predicts Pseudomonas aeruginosa virulence in lung infections; Sawa T et al.; The role of quorum sensing by Pseudomonas aeruginosa in producing cytotoxicity has not been fully investigated . Strains of P . aeruginosa have been characterized as having an invasive or a cytotoxic phenotype (S . M . J . Fleiszig et al., Infect . Immun . 65:579-586, 1997) . We noted that the application of a large inoculum of the invasive strain 6294 caused cytotoxicity of cultured epithelial cells . To investigate this dose-related cytotoxicity, we compared the behavior of 6294 to that of another invasive strain, PAO1, and determined whether the cytotoxicity could be related to quorum sensing . Both invasive strains, 6294 and PAO1, appear to have quorum-sensing systems that were operative when large doses of bacteria were applied to cultured lung epithelial cells or instilled into the lungs of animals . Nonetheless, only 6294 was cytotoxic . Cytotoxicity induced by 6294 correlated with increased elastase production . These experiments suggest that there are multiple mechanisms for the induction of cytotoxicity, pathology, and mortality in vivo . However, in vivo cytotoxicity and mortality, but not pathology, could be predicted by quantitative in vitro cellular damage experiments utilizing a range of bacteria-to-cell ratios . It appears that quorum sensing may inversely correlate with virulence in that strains that produced PAI {N-(3-oxododecanoyl) homoserine lactone} also appeared to attract more polymorphonuclear leukocytes in vivo and were possibly eliminated more quickly . In addition, exoproduct production in bacteriological medium in vitro may differ significantly from exoproduct expression from infections in vivo or during cocultivation of bacteria with tissue culture cells. Infect Immun, 1998 Jul, 66(7), 3164 - 9 Role of alveolar macrophages in initiation and regulation of inflammation in Pseudomonas aeruginosa pneumonia; Kooguchi K et al.; To evaluate the role of alveolar macrophages (AMs) in acute Pseudomonas aeruginosa pneumonia in mice, AMs were depleted by aerosol inhalation of liposomes containing clodronate disodium . AM-depleted mice were then intratracheally infected with 5 x 10(5) CFU of P . aeruginosa . In addition to monitoring neutrophil recruitment and chemokine releases, lung injury was evaluated soon after infection (8 h) and at a later time (48 h) . At 8 h, depletion of AMs reduced neutrophil recruitment, chemokine release, and lung injury . At 48 h, however, depletion of AMs decreased bacterial clearance and resulted in delayed movement of neutrophils from the site of inflammation with aggravated lung injury . With instillation of 5 x 10(7) CFU of bacteria, AM-depleted mice showed low mortality within 24 h of infection but high mortality at a later time, in contrast to non-AM-depleted mice . These results demonstrate that depletion of AMs has beneficial early effects but deleterious late effects on lung injury and survival in cases of P . aeruginosa pneumonia. Infect Immun, 1998 Jul, 66(7), 3072 - 9 Pseudomonas aeruginosa exoenzyme S is a mitogen but not a superantigen for human T lymphocytes; Bruno TF et al.; Virtually all cystic fibrosis (CF) patients become infected with Pseudomonas aeruginosa, and once the infection is established, the organism is rarely cleared . One of the P . aeruginosa virulence factors, exoenzyme S, has been shown to correlate with increased morbidity and mortality both in rat models of chronic pulmonary inflammation and in human CF patients . It has previously been shown that exoenzyme S is a potent stimulus for the proliferation of T cells in greater than 95% of adults, which could contribute to the pathogenesis of CF . The goal of this study was to determine the mechanism of T-cell stimulation by exoenzyme S in an effort to shed light on the immune response and contribute to understanding its role in P . aeruginosa pathogenesis . The current studies demonstrate that exoenzyme S stimulates naive T cells, since fetal blood lymphocytes proliferated and adult lymphocytes that expressed CD45RA proliferated . The percentage of T cells activated by exoenzyme S after a 4-h culture (as measured by CD69 surface expression) was intermediate in magnitude compared to levels induced by a panel of superantigens and mitogens . To determine the mechanism of activation, the requirement for accessory cells was investigated . The proliferative response to exoenzyme S was dependent on the presence of accessory cells but was not blocked by an anti-DR antibody . Exoenzyme S activated both CD4(+) and CD8(+) T cells, but CD4(+) T cells were preferentially activated . The Vbeta repertoire of donor T cells showed no preferential activation or preferential expansion after stimulation by exoenzyme S, suggesting that it is not a superantigen . Taken together, our data suggest that exoenzyme S is a T-cell mitogen but not a superantigen . Activation of a large percentage of T lymphocytes by exoenzyme S may produce a lymphocyte-mediated inflammatory response that should be considered in the pathogenesis of CF. Am J Respir Crit Care Med, 1998 Jun, 157(6 Pt 1), 1764 - 9 Circulating immunoreactive interleukin-6 in cystic fibrosis; Nixon LS et al.; We measured circulating and sputum-sol concentrations of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), neutrophil elastase-alpha1-antiproteinase complex (NEAPC), and C-reactive protein (CRP) in an exacerbation, after antibiotic treatment, and in clinically stable patients with cystic fibrosis and chronic pulmonary infection with Pseudomonas aeruginosa . The aim was to determine the compartmental patterns of a proinflammatory and anti-inflammatory cytokine compared with other markers of inflammatory activity in cystic fibrosis . IL-6, NEAPC, CRP, and absolute neutrophil count were reduced after antibiotic treatment, p < 0.01 . IL-6 and CRP concentrations were greater, p = 0.007, and p = 0.01, respectively, in a stable group of patients compared with those at the end of an exacerbation . IL-6 and CRP concentrations were related (r = 0.836, p < 0.0001), and both were greater than in matched control subjects (p < 0.001) at all times studied . Sputum-sol concentrations of IL-6 after treatment were positively related to FEV1 and FVC and inversely related to concentrations of neutrophil elastase . The separation between patients and healthy subjects, and the reduction of IL-6 after antibiotic treatment indicates it could be used as a marker of inflammation, but its relationship to other markers depends on the compartment in which it is measured. Am J Physiol, 1998 Jun, 274(6 Pt 1), L893 - 900 Pseudomonas pyocyanine alters calcium signaling in human airway epithelial cells; Denning GM et al.; Pseudomonas aeruginosa, an opportunistic human pathogen, causes both acute and chronic lung disease . P . aeruginosa exerts many of its pathophysiological effects by secreting virulence factors, including pyocyanine, a redox-active compound that increases intracellular oxidant stress . Because oxidant stress has been shown to affect cytosolic Ca2+ concentration ({Ca2+}c) in other cell types, we studied the effect of pyocyanine on {Ca2+}c in human airway epithelial cells (A549 and HBE) . At lower concentrations, pyocyanine inhibits inositol 1,4,5-trisphosphate formation and {Ca2+}c increases in response to G protein-coupled receptor agonists . Conversely, at higher concentrations, pyocyanine itself increases {Ca2+}c . The pyocyanine-dependent {Ca2+}c increase appears to be oxidant dependent and to result from increased inositol trisphosphate and release of Ca2+ from intracellular stores . Ca2+ plays a central role in epithelial cell function, including regulation of ion transport, mucus secretion, and ciliary beat frequency . By disrupting Ca2+ homeostasis, pyocyanine could interfere with these critical functions and contribute to the pathophysiological effects observed in Pseudomonas-associated lung disease. Biotechnology (N Y), 1996 Feb, 14(2), 203 - 8 Development of new cloning vectors for the production of immunogenic outer membrane fusion proteins in Escherichia coli; Cornelis P et al.; The Pseudomonas aeruginosa lipoprotein gene (oprI) was modified by cloning an in-frame polylinker in both orientations at the end of oprI . The resulting plasmids pVUB1 and pVUB2 allow high lipoprotein production in E . coli after IPTG induction . The modified lipoproteins are present in the outer membrane and surface-exposed . Outer membrane-bound fusion proteins of different sizes were produced and used to generate antibodies without use of adjuvant . An 87 bp DNA fragment from the vp72 capsid protein gene of African Swine Fever virus (ASFV) and the entire Leishmania major glycoprotein gp63 gene were expressed in this system . Finally, a fusion lipoprotein containing a 16 amino acid epitope from the pre-S2b region of Hepatitis B virus (HBV) was presented by an antigen-presenting cell line to a T-cell hybridoma while the corresponding cross-linked S2b peptide was not . The results suggest that OprI-based fusion proteins can be used to generate both humoral and cellular immune responses. APMIS, 1998 Apr, 106(4), 456 - 62 Ribotyping of Pseudomonas aeruginosa from infected patients: evidence of common strain types; Ferrus MA et al.; Sixty-nine strains of Pseudomonas aeruginosa isolated from infected patients at three hospitals in Valencia were serotyped and analyzed by comparison of restriction fragment length polymorphisms of ribosomal RNA genes (ribotyping) . Genomic Southern blots of EcoRI restriction digests were hybridized with a universal rRNA gene probe from Escherichia coli 16S and 23S rRNA . Strains were genetically diverse and 12 different ribotypes of 2 to 7 bands between 5 and 21.5 kb were defined . All strains shared a common band of 6.0 kb . The predominant ribotypes were R1 and R2, representing 25% and 41% of all isolates, respectively . Ribotypes were not consistently associated with serotypes, but they clearly subtyped strains of the same serotype . This study demonstrated the prevalence of certain strain types associated with infected patients at Valencia hospitals, confirming the high typability and reproducibility of a single enzyme ribotyping for epidemiological studies of Pseudomonas aeruginosa . Ribotyping could be particularly useful if used in conjunction with serotyping. Clin Infect Dis, 1998 Jun, 26(6), 1440 - 6 Something's rotten: a nosocomial outbreak of malodorous Pseudomonas aeruginosa; Labarca JA et al.; From July 1994 through November 1996, a phenotypically unique strain of Pseudomonas aeruginosa producing a pungent, "rotten-potato" odor and a positive lysine decarboxylase reaction was isolated from 39 patients at UCLA Medical Center (Los Angeles) . Most cases (95%) were in intensive care units and had clinical infections (72%) . Most isolates (74%) were recovered from cultures of respiratory secretions . To determine risk factors for acquisition of the organism, 23 cases were compared with 23 randomly selected controls matched by service and isolate date . Multivariate analysis revealed that isolation of malodorous P . aeruginosa was associated with mechanical ventilation of > 24 hours' duration (odds ratio {OR} = 9.4; P = .001) and transfer from an outside hospital (OR = 5.7; P = .04) . DNA from outbreak strains hybridized to P . aeruginosa-specific toxin A and phospholipase C gene probes and all outbreak isolates tested were found to be identical by use of pulsed-field gel electrophoresis . An unusual phenotypic characteristic of the strain led to the recognition of a nosocomial outbreak of P . aeruginosa infection associated with mechanical ventilation. Biochem Biophys Res Commun, 1998 Jun 9, 247(1), 142 - 5 Identification of the catalytic triad of the protein D2 protease in Pseudomonas aeruginosa; Yoshihara E et al.; We reported recently that protein D2 (OprD) porin of Pseudomonas aeruginosa bears protease activity (FEBS Letters 394, 179-182, 1996) . To identify the catalytic residues of OprD, we introduced the site-directed mutations replacing the putative catalytic triad His156, Asp208, and Ser296 with glutamine, asparagine, and alanine, respectively . The OprD proteins purified from the chromosomal oprD-deficient mutants harboring the plasmids encoding the site-directed mutations showed protease activity less than 0.1% of that of the wild-type OprD . These site-directed mutageneses caused undetectable changes in the pore-forming activity of OprD as measured by single-channel conductance by the planar lipid bilayer . The minimum inhibitory concentration of imipenem in mutants having the replaced catalytic triads was identical with that in the wild-type strain . On the other hand, introduction of the mutation at His367 replacing with glutamine, the site that is supposed to be unrelated to the catalytic sites, showed the unchanged protease activity . These results unequivocally demonstrate that OprD is the protease bearing porin and catalyzes the reaction at His156, Asp208, and Ser296 residues. Biochemistry, 1998 Jun 16, 37(24), 8659 - 73 Determination of the magnetic axes of cobalt(II) and nickel(II) azurins from 1H NMR data: influence of the metal and axial ligands on the origin of magnetic anisotropy in blue copper proteins; Donaire A et al.; The orientation and the axial, Deltachiax, and rhombic, Deltachirh, components of the magnetic susceptibility tensor anisotropy for the cobalt(II) and nickel(II) derivatives of azurin from Pseudomonas aeruginosa have been determined from 1H NMR data . For both derivatives, the axial geometry of the system determines the orientation of the chi-tensor, whose z-axis forms an angle of 18.6 and 20.1 degrees with the Cu-OGly45 axial bond in the cobalt(II) and nickel(II) derivatives, respectively . For protons close to this axis, large negative pseudocontact shifts are observed, while those close to the NNS plane of the equatorial ligands experience lower and positive pseudocontact shifts for the same distance . Dipolar shifts are larger in the cobalt derivative, not only because of the larger spin number but also due to its intrinsically higher anisotropy . The contact contribution to the hyperfine shifts for the coordinated residues has been evaluated and analyzed in terms of unpaired spin delocalization mechanisms and geometry considerations . The results are extended to other blue copper proteins whose cobalt derivatives have been studied by 1H NMR . The electronic structure and its implications in the redox properties of the native copper proteins are also commented. BMJ, 1998 Jun 13, 316(7147), 1771 - 5 Clinical outcome in relation to care in centres specialising in cystic fibrosis: cross sectional study; Mahadeva R et al.; OBJECTIVES: To assess the effect on clinical outcome of managing paediatric and adult patients with cystic fibrosis at specialised cystic fibrosis centres . DESIGN: Cross sectional study . SETTING: Two adult cystic fibrosis centres in the United Kingdom . SUBJECTS: Patients from an adult cystic fibrosis centre in Manchester were subdivided into those who had received continuous care from paediatric and adult cystic fibrosis centres (group A), and those who had received paediatric care in a centre not specialising in cystic fibrosis followed by adult care in a cystic fibrosis centre (group B) . Group C were referrals to the new adult cystic fibrosis centre in Cambridge who had received neither paediatric nor adult centre care for their cystic fibrosis . MAIN OUTCOME MEASURES: Body mass index (weight (kg)/height (m2)), lung function (forced expiratory volume in one second (FEV1 percentage of predicted)), the Northern chest x ray film score, and age at colonisation with Pseudomonas aeruginosa . RESULTS: A prominent stepwise increase in body mass index was associated with increasing amounts of care at a cystic fibrosis centre; 18.3, 20.2, and 21.3 for groups C, B, and A respectively (P<0.001) . Improved nutritional status was correlated with a higher FEV1 and better (lower) chest x ray film scores; r=0 . 52 and -0.45 respectively (P<0.001 for both) . CONCLUSION: These findings provide the first direct evidence that management of cystic fibrosis in paediatric and adult cystic fibrosis centres results in a better clinical outcome, and strongly supports the provision of these specialist services. Pediatr Pulmonol, 1998 May, 25(5), 304 - 8 Stenotrophomonas maltophilia in cystic fibrosis: incidence and prevalence; Demko CA et al.; Stenotrophomonas maltophilia (SM) was recovered from 211 of 773 cystic fibrosis (CF) patients followed for at least one year, and seen between 1982 and 1994 . Yearly prevalence (5.6% to 8.7%) and incidence rates (1.6% to 5.7%) showed no trends . SM persistence varied greatly and was unlike that of Pseudomonas aeruginosa . Fifty percent of SM-positive patients had only one positive culture and only 24 (11%) remained chronically infected . Although SM-positive patients were more likely to be hospitalized than SM-negative patients, for 55% of SM-positive patients, acquisition did not appear to follow hospitalization . Of 40 SM-positive patients who had a CF sibling, only 10 siblings were ever culture positive . When stratified by FEV1, the two-year survival for SM-positive with mild/moderate disease (98%) and severe disease (78%) was similar to that of our SM-negative patients . Five-year survival was only 40% for SM-positive patients with initially severe pulmonary status, compared with 72% for the SM-negative patients . Seventy percent of the original SM isolates were panresistant (susceptible to no more than one antimicrobial agent) . Ten years later, panresistance was 84% . Despite our reassuring experience with SM, including lack of sibling concordance, the fact that the majority of our patients had no hospital exposure prior to acquisition, the high incidence of transient infection, and the seemingly unaffected two-year survival, there are insufficient data to definitively conclude that segregation of these patients would be beneficial . The increasing prevalence of multiply resistant gram-negative pathogens in CF patients suggests the need for continued caution with any panresistant pathogen. J Biol Chem, 1998 Jun 26, 273(26), 16305 - 10 14-3-3zeta binds a phosphorylated Raf peptide and an unphosphorylated peptide via its conserved amphipathic groove; Petosa C et al.; 14-3-3 proteins bind a variety of molecules involved in signal transduction, cell cycle regulation and apoptosis . 14-3-3 binds ligands such as Raf-1 kinase and Bad by recognizing the phosphorylated consensus motif, RSXpSXP, but must bind unphosphorylated ligands, such as glycoprotein Ib and Pseudomonas aeruginosa exoenzyme S, via a different motif . Here we report the crystal structures of the zeta isoform of 14-3-3 in complex with two peptide ligands: a Raf-derived phosphopeptide (pS-Raf-259, LSQRQRSTpSTPNVHMV) and an unphosphorylated peptide derived from phage display (R18, PHCVPRDLSWLDLEANMCLP) that inhibits binding of exoenzyme S and Raf-1 . The two peptides bind within a conserved amphipathic groove on the surface of 14-3-3 at overlapping but distinct sites . The phosphoserine of pS-Raf-259 engages a cluster of basic residues (Lys49, Arg56, Arg60, and Arg127), whereas R18 binds via the amphipathic sequence, WLDLE, with its two acidic groups coordinating the same basic cluster . 14-3-3 is dimeric, and its two peptide-binding grooves are arranged in an antiparallel fashion, 30 A apart . The ability of each groove to bind different peptide motifs suggests how 14-3-3 can act in signal transduction by inducing either homodimer or heterodimer formation in its target proteins. Alcohol Alcohol, 1998 May-Jun, 33(3), 273 - 80 Characteristics of aldehyde dehydrogenases of certain aerobic bacteria representing human colonic flora; Nosova T et al.; We have proposed the existence of a bacteriocolonic pathway for ethanol oxidation resulting in high intracolonic levels of toxic and carcinogenic acetaldehyde . This study was aimed at determining the ability of the aldehyde dehydrogenases (ALDH) of aerobic bacteria representing human colonic flora to metabolize intracolonically derived acetaldehyde . The apparent Michaelis constant (Km) values for acetaldehyde were determined in crude extracts of five aerobic bacterial strains, alcohol dehydrogenase (ADH) and ALDH activities of these bacteria at conditions prevailing in the human large intestine after moderate drinking were then compared . The effect of cyanamide, a potent inhibitor of mammalian ALDH, on bacterial ALDH activity was also studied . The apparent Km for acetaldehyde varied from 6.8 (NADP+-linked ALDH of Escherichia coli IH 13369) to 205 microM (NAD+-linked ALDH of Pseudomonas aeruginosa IH 35342), and maximal velocity varied from 6 nmol/min/mg (NAD+-linked ALDH of Klebsiella pneumoniae IH 35385) to 39 nmol/min/mg (NAD+-linked ALDH of Pseudomonas aeruginosa IH 35342) . At pH 7.4, and at ethanol and acetaldehyde concentrations that may be prevalent in the human colon after moderate drinking, ADH activity in four out of five bacterial strains were 10-50 times higher than their ALDH activity . Cyanamide inhibited only NAD+-linked ALDH activity of Pseudomonas aeruginosa IH 35342 at concentrations starting from 0.1 nmM . We conclude that ALDHs of the colonic aerobic bacteria are able to metabolize endogenic acetaldehyde . However, the ability of ALDHs to metabolize intracolonic acetaldehyde levels associated with alcohol drinking is rather low . Large differences between ADH and ALDH activities of the bacteria found in this study may contribute to the accumulation of acetaldehyde in the large intestine after moderate drinking . ALDH activities of colonic bacteria were poorly inhibited by cyanamide . This study supports the crucial role of intestinal bacteria in the accumulation of intracolonic acetaldehyde after drinking alcohol . Individual variations in human colonic flora may contribute to the risk of alcohol-related gastrointestinal morbidity. J Antimicrob Chemother, 1998 May, 41(5), 557 - 61 Application of X-ray micrography and imaging to study the effect of gentamicin on Pseudomonas aeruginosa; Rajyaguru JM et al.; Aminoglycosides disrupt the outer membrane of Pseudomonas aeruginosa to facilitate access to their intracellular target . High-resolution X-ray micrography of live specimens is a relatively new technique . We used laser (nanosecond) plasma to image live cells of P . aeruginosa ATCC 27853 . After exposure to 25 mg/L gentamicin for 15 min . we observed perturbation of the cell surface, membrane blebbings (370 nm and 273 nm diameter) away from the cell, formation of distinct channels (241 nm long) resulting from indentation and induction of cell elongation from 3-3.6 microm (control) to 4.6-5.26 microm (gentamicin-treated cells) . These data illustrate the potential of high-resolution X-ray micrography for studying effects of drugs on live microbiological specimens. Biotechnol Prog, 1998 May, 14(3), 534 - 6 Biodegradability behavior of cross-linked calcium caseinate films Mezgheni E, Vachon C, Lacroix M. gamma-Irradiation was used to produce free-standing biodegradable caseinate films . The effect of the irradiation dose (i.e., the number of cross-links) on the biodegradability behavior of these films using a strain of Pseudomonas aeruginosa was investigated . Results showed that the overall degradation processes were similar for both types of films (4 or 64 kGy) . The main difference was observed in terms of the period of degradation which was delayed 8 days for the film containing the highest extent of cross-linking (64 kGy). J Clin Invest, 1998 Jun 1, 101(11), 2598 - 605 Activation of NF-kappaB by adherent Pseudomonas aeruginosa in normal and cystic fibrosis respiratory epithelial cells; DiMango E et al.; PMN-dominated airway inflammation is a major component of cystic fibrosis (CF) lung disease . Epithelial cells respond to organisms such as Pseudomonas aeruginosa, the major pathogen in CF, by expressing the leukocyte chemokine IL-8 . Experiments were performed using several different types of respiratory epithelial cells that demonstrate that ligation of ceramide-associated receptors on epithelial surfaces by P . aeruginosa pili is a major stimulus for the translocation of transcription factor nuclear factor (NF)-kappaB and initiation of IL-8 expression by epithelial cells . Using electrophoretic mobility shift assays and Western hybridizations, nuclear NF-kappaB was found shortly after epithelial cells were stimulated by either whole organisms, isolated pili, or antibody to the pilin receptor asialoGM1 . IB3 cells, which express mutations in cystic fibrosis transmembrane conductance regulator (CFTR) (DeltaF508/W1282X), were noted to have significantly greater amounts of endogenous nuclear NF-kappaB, but not the transcription factor C/EBP, than CF cells corrected by episomal copies of normal CFTR (C-38) or IB3 cells grown at a permissive temperature (25 degreesC) . Activation of NF-kappaB and subsequent IL-8 expression in epithelial cells can result from activation of at least two pathways: an exogenous signaling cascade that is activated by ligation of ceramide-associated adhesins such as P . aeruginosa pilin, or endogenous stimulation, suggested to be a consequence of cell stress caused by the accumulation of mutant CFTR in the endoplasmic reticulum. Antimicrob Agents Chemother, 1998 Jun, 42(6), 1503 - 5 Postantibiotic effect of trovafloxacin against gram-positive and -negative organisms; Pankuch GA et al.; Trovafloxacin pneumococcal and staphylococcal postantibiotic effects (PAEs) were 0.7 to 1.8 and 0.7 to 2.4 h, respectively . For Escherichia coli and Pseudomonas aeruginosa, PAEs were 2.4 to 4.4 h . Pneumococcal and staphylococcal postantibiotic sub-MIC effects (PA-SMEs) (0.4 times the MIC) were 2.3 to 3.7 and 2.4 to > 9.2 h, respectively, and E . coli PA-SMEs (0.3 times the MIC) were 6.8 to > 12.0 h . For one P . aeruginosa strain, the PA-SME (0.4 times the MIC) was > 10 h; in the other, rapid bactericidal activity precluded measurement. Antimicrob Agents Chemother, 1998 Jun, 42(6), 1365 - 9 Changes in MIC alter responses of Pseudomonas aeruginosa to tobramycin exposure; Ioannides-Demos LL et al.; The pharmacokinetic parameters determining antibiotic efficacy are peak concentrations (Cmax), minimum (trough) concentrations (Cmin), and area under the concentration-time curve (AUC) . There is general agreement about the importance of Cmax and AUC for aminoglycosides, but this is not so for maintenance of Cmin . With in vitro exposures modelling in vivo administration, Pseudomonas aeruginosa reference strain ATCC 27853 (MIC, 1 mg/liter) and a higher-MIC (relatively resistant) clinical isolate (MIC, 4 mg/liter) were used to explore bacteriostatic and bactericidal outcomes . With P . aeruginosa ATCC 27853, kill followed a complete bolus profile with a 30-min postdistribution peak (Cpeak30) of 10 mg/liter . The clinical isolate required a Cpeak30 bolus profile of 20 mg/liter for kill, and there was no difference between the efficacies of the bolus and infusion exposures . Bolus profiles that were truncated at 8.5 h and producing sublethal effects were then combined with a wide range of Cmins . With a Cpeak30 profile of 8 mg/liter, P . aeruginosa ATCC 27853 showed a graded bacteriostatic response until a Cmin of > or = 0.8 mg/liter, when complete kill resulted . In contrast, bactericidal effects on the clinical isolate required a Cpeak30 profile of 18 mg/liter with a Cmin of > or = 1.0 mg/liter . Therefore, Cmin also contributes to the bactericidal effect of tobramycin, with requirements showing minor variation with change in MIC . Dosing principles for relatively resistant (higher-MIC) organisms are suggested from the data . Relatively higher aminoglycoside doses via infusion regimens are likely to be needed to generate higher peak concentrations and higher AUC values necessary for bactericidal effect in resistant organisms . Maintenance of trough concentrations on the order of 1.0 mg/liter during the interdose interval will tend to guard against the possibility of inadequate peak and AUC exposures for kill. Vestn Oftalmol, 1998 Mar-Apr, 114(2), 13 - 7 {Emergency penetrating keratoplasty in infective lesions of the cornea at the site of sutures}; Kasparov AA et al.; Urgent perforating keratoplasty was carried out in 52 patients with infective involvement of the cornea at the site of sutures after cataract extraction, perforating keratoplasty, and treatment of penetrating wounds of the cornea . The inflammation was most often caused by Pseudomonas aeruginosa (32%), cocci (33.6%), and fungi (14.1%) . The technique of perforating keratoplasty for cases with different localization and extent of purulent infiltration is described and results of pathohistological examination of removed disks presented . The authors emphasize the importance of timely removal of monofilament sutures and thorough care of sutured sites . Urgent perforating keratoplasty arrested acute inflammations in 90% of cases and preserved or improved visual acuity in 70% of cases. Kansenshogaku Zasshi, 1998 Apr, 72(4), 395 - 409 {Study on pathogenetic role of myeloperoxidase, tumor necrosis factor alpha, interferon gamma in chronic airway infection with Pseudomonas aeruginosa}; Takeda H; This study was performed to demonstrate the pathogenetic role of mucoid-alginate produced by Pseudomonas aeruginosa in relation to immuno-interaction in host cells . The obtained results are as follows . 1 . Clinical Observation The concentration of MPO, TNF alpha, and IFN gamma in peripheral blood of 37 cases of chronic airway infection were observed . The cases were divided into the groups with mucoid strain positive (21 cases) and negative (16 cases), and each group was also divided into subgroups with active clinical symptom and without . High concentration of serum IFN gamma was shown in the group of mucoid strain positive cases (0.10 +/- 0.08 IU/ml in healthy volunteers group, 0.54 +/- 0.56 IU/ml in mucoid group with symptom: p < 0.01, 0.09 +/- 0.15 IU/ml in mucoid group without symptom, 0.13 +/- 0.13 IU/ml in non-mucoid group with symptom, 0.09 +/- 0.13 IU/ml in non-mucoid group without symptom) . But plasma concentrations of MPO and serum concentrations of TNF alpha were not increased in all groups, except MPO in non-mucoid group with symptom . It indicates that IFN gamma has a characteristic role in chronic airway infection with mucoid Pseudomonas aeruginosa (P . aeruginosa) . 2 . Experimental Observation One hundred twenty-two of alginate-immunized mice were made by 5 times of intraabdominal injection with purified alginate extracted from mucoid P . aeruginosa PT-1252 . 4 x 10(6) CFU/body of mucoid P . aeruginosa PT-1252 or 20 micrograms/body of purified alginate was intubated per trachea into the immunized mice, and changes in mean fluorescence intensity (MFI) of MPO and TNF alpha on neutrophils and IFN gamma on CD3 positive lymphocytes in BALF were observed . MFI of MPO (99.7 +/- 16.5-->177.0 +/- 23.1: p < 0.001) and TNF alpha (59.2 +/- 13.3-->197.0 +/- 62.0: p < 0.001) were increased on day 2 and they were kept up till on day 30 in P . aeruginosa group . But in alginate group they were temporally decreased on day 2, and they were gradually increased (MPO: 99.7 +/- 16.5-->212.6 +/- 12.1: p < 0.001, TNF alpha: 59.2 +/- 13.3-->162.4 +/- 30.9: p < 0.01) . The temporally decrease of MPO and TNF alpha on day 2 in alginate group may suggest that a large amount of alginate inhibits neutrophils chemotaxis . MFI of IFN gamma in P . aeruginosa was also increased (110.0 +/- 32.9-->198.3 +/- 23.0: p < 0.01) on day 2 and it was gradually decreased on day 30 (156.0 +/- 13.8) . In alginate group, MFI of IFN gamma in BALF was increased on day 5 (110.0 +/- 32.9-->201.0 +/- 49.3: p < 0.001) and kept a high level till day 30 (223.0 +/- 57.5: p < 0.01) . In morphological, the moderate grade of lymphocytes infiltration observed in intersitial area of lung tissue on both groups of P . aeruginosa and alginate intubation mice . The findings was continuously seen from day 5 through day 30 . The lymphocytes infiltration appeared in lung tissue can be though as an result of immuno-interaction with IFN gamma positive lymphocytes and mucoid-alginate . Inferences are drawn from the clinical results and the experimental ones that the CD3 positive IFN gamma lymphocytes expressed by immunological interaction with mucoid-alginate and the IFN gamma lymphocytes (Th1 like cells) plays an important role on advancement of chronic airway infection with mucoid P . aeruginosa. Kansenshogaku Zasshi, 1998 Apr, 72(4), 379 - 94 {Study on the role of mucoid strain in chronic airway infections}; Sakagawa E; This study was investigated to eludiate the clinical role of immune complexes (IC) formed by alginate of persistently colonized mucoid strain in chronic airway infection . 1 . Clinical Observation Twenty-four cases colonized with mucoid strain and 16 cases with non-mucoid strain were enrolled . Their serum indexes to anti-alginate specific IgG subclass antibodies and level of IC were determined by enzyme linked immunosorbent assays . Significantly higher level of the IgG1, IgG3 and higher level of IC were observed in the group of mucoid positive cases . Deposition of IgG1, IgG3, and IC on the affected part of interstitial lung tissue in a case of persistent infection with mucoid Pseudomonas aeruginosa was detected . 2 . Experimental Observation Ninety mice immunized with alginate were used . They were intubated with 20 micrograms/body of alginate, 4 x 10(6) colony forming unit/body of mucoid Pseudomonas aeruginosa: PT1252, or 40 microliters/body of IC into each group . Then after Fc gamma receptor (Fc gamma R) on neutrophils in bronchoalveolar lavage fluid was analyzed by flow cytometric technique . Expression of Fc gamma R on neutrophils was inhibited against alginate intubation and IC one . In contrast, Fc gamma R expression was increased at initial phase after mucoid Pseudomonas aeruginosa intubation, which was influenced by nonspecific activation of neutrophils due to bacterial infection . 3 . Conclusive Words From the above, The expression of Fc gamma R on neutrophils was inhibited by alginate . It may be that IC deposition on lung tissue in chronic airway infection with mucoid strain was persisted by poor binding ability of neutrophils to IC, in spite of a large numbers of neutrophils accumulation . Consequently, the immune-reaction induced by alginate makes clinical prognosis of such patients more intractable. J Bacteriol, 1998 Jun, 180(12), 3209 - 17 Cloning and comparison of fliC genes and identification of glycosylation in the flagellin of Pseudomonas aeruginosa a-type strains; Brimer CD et al.; Pseudomonas aeruginosa a-type strains produce flagellin proteins which vary in molecular weight between strains . To compare the properties of a-type flagellins, the flagellin genes of several Pseudomonas aeruginosa a-type strains, as determined by interaction with specific anti-a monoclonal antibody, were cloned and sequenced . PCR amplification of the a-type flagellin gene fragments from five strains each yielded a 1.02-kb product, indicating that the gene size is not likely to be responsible for the observed molecular weight differences among the a-type strains . The flagellin amino acid sequences of several a-type strains (170,018, 5933, 5939, and PAK) were compared, and that of 170,018 was compared with that of PAO1, a b-type strain . The former comparisons revealed that a-type strains are similar in amino acid sequence, while the latter comparison revealed differences between 170,018 and PAO1 . Posttranslational modification was explored for its contribution to the observed differences in molecular weight among the a-type strains . A biotin-hydrazide glycosylation assay was performed on the flagellins of three a-type strains (170,018, 5933, and 5939) and one b-type strain (M2), revealing a positive glycosylation reaction for strains 5933 and 5939 and a negative reaction for 170,018 and M2 . Deglycosylation of the flagellin proteins with trifluoromethanesulfonic acid (TFMS) confirmed the glycosylation results . A molecular weight shift was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis for the TFMS-treated flagellins of 5933 and 5939 . These results indicate that the molecular weight discrepancies observed for the a-type flagellins can be attributed, at least in part, to glycosylation of the protein . Anti-a flagellin monoclonal antibody reacted with the TFMS-treated flagellins, suggesting that the glycosyl groups are not a necessary component of the epitope for the human anti-a monoclonal antibody . Comparisons between a-type sequences and a b-type sequence (PAO1) will aid in delineation of the epitope for this monoclonal antibody. J Appl Physiol, 1998 Jun, 84(6), 1991 - 9 Pyridoxalated hemoglobin polyoxyethylene conjugate reverses hyperdynamic circulation in septic sheep; Bone HG et al.; We investigated the effects of modified hemoglobin on regional blood flow and function of different organs during hyperdynamic sepsis . Fourteen sheep were surgically prepared for the study . After a 5-day recovery period, a continuous infusion of live Pseudomonas aeruginosa bacteria was begun and maintained for 48 h . At 24 h, after a hyperdynamic circulation had developed, the animals were randomly assigned to two groups: 1) a treatment group (n = 7) that received an infusion with 100 mg/kg pyridoxalated hemoglobin polyoxyethylene conjugate (PHP) over 30 min and 2) a control group (n = 7) that received only the vehicle . PHP infusion increased mean arterial pressure from 86 +/- 2.8 to 101.8 +/- 3.5 mmHg (P < 0.05) and systemic vascular resistance index from 769 +/- 42.1 to 1,087 +/- 56.8 dyn . s . m2 . cm-5 (P < 0.05) . PHP infusion did not decrease regional blood flow, measured with fluorescent microspheres, below the baseline values in any of the analyzed tissues . None of the investigated blood chemistry variables showed any changes indicative of impaired organ function after PHP infusion . In our model of ovine sepsis we found no side effects after PHP infusion that would limit the use of PHP as a nitric oxide scavenger in sepsis. J Biol Chem, 1998 Jun 5, 273(23), 14368 - 73 Purification and characterization of a novel ceramidase from Pseudomonas aeruginosa; Okino N et al.; We report here a novel type of ceramidase of Pseudomonas aeruginosa AN17 isolated from the skin of a patient with atopic dermatitis . The enzyme was purified 83,400-fold with an overall yield of 21.1% from a culture supernatant of strain AN17 . After being stained with a silver staining solution, the purified enzyme showed a single protein band, and its molecular mass was estimated to be 70 kDa on SDS-polyacrylamide gel electrophoresis . The enzyme showed quite wide specificity for various ceramides, i.e . it hydrolyzed ceramides containing C12:0-C18:0 fatty acids and 7-nitrobenz-2-oxa-1, 3-diazole-labeled dodecanoic acid, and not only ceramide containing sphingosine (d18:1) or sphinganine (d18:0) but also phytosphingosine (t18:0) as the long-chain base . However, the enzyme did not hydrolyze galactosylceramide, sulfatide, GM1, or sphingomyelin, and thus was clearly distinguished from a Pseudomonas sphingolipid ceramide N-deacylase (Ito, M., Kurita, T., and Kita, K . (1995) J . Biol . Chem . 270, 24370-24374) . This bacterial ceramidase had a pH optimum of 8.0-9.0, an apparent Km of 139 microM, and a Vmax of 5.3 micromol/min/mg using N-palmitoylsphingosine as the substrate . The enzyme appears to require Ca2+ for expression of the activity . Interestingly, the 70-kDa protein catalyzed a reversible reaction in which the N-acyl linkage of ceramide was either cleaved or synthesized . Our study demonstrated that ceramidase is widely distributed from bacteria to mammals. Arch Dermatol Res, 1998 Apr, 290(4), 211 - 4 Lipoteichoic acid and protein-A from Staphylococcus aureus stimulate release of hepatocyte growth factor (HGF) by human dermal fibroblasts; Baroni A et al.; In this study we demonstrated that Staphylococcus aureus lipoteichoic acid (LTA) and protein-A (PA) induce the release from human dermal fibroblasts of hepatocyte growth factor (HGF), a proliferation factor of epithelial cells (including keratinocytes) . In contrast, LPS and porins from Pseudomonas aeruginosa did not stimulate HGF production . Recombinant human IL-1 beta induced HGF release . This production was synergistically enhanced when in association with LTA (by more than twice) and PA (by about two-thirds) . Controls were performed in the presence of bacterial components alone . In previous studies we have shown that LPS and porins are inducers of IL-1 alpha and beta and other cytokines from human monocytes . Therefore it is possible that in inflammatory cutaneous foci and infected wounds, bacterial components may induce HGF release from dermal human fibroblasts . LTA and PA act directly, while LPS and porins act indirectly, through the release of cytokines by monocytes/macrophages . HGF plays an important role in the repair of cutaneous tissue during gram-positive and gram-negative infections. Gene Ther, 1998 Mar, 5(3), 345 - 51 Effects of bronchopulmonary inflammation induced by pseudomonas aeruginosa on adenovirus-mediated gene transfer to airway epithelial cells in mice; van Heeckeren A et al.; Cystic fibrosis (CF) patients have endobronchial inflammation caused by infection with mucoid Pseudomonas aeruginosa . Since adenovirus vectors are being studied for gene therapy for CF, we sought to determine whether bronchopulmonary inflammation would influence adenovirus-mediated gene transfer . We hypothesized that bronchopulmonary inflammation in mice inoculated with mucoid P . aeruginosa would be associated with a decrease in the efficacy of adenovirus-mediated gene transfer . Agarose beads embedded with mucoid P . aeruginosa (6 x 10(4) c.f.u . per mouse) were inoculated transtracheally into C57BL/6 mice . Control mice received sterile agarose beads . Ten days after inoculation with agarose beads, recombinant adenovirus containing the beta-galactosidase reporter gene (Ad2/beta Gal-2) was administered intranasally (1.1 x 10(9) IU per mouse), and mice were killed 3 days later . The extent of inflammation, determined by neutrophil numbers in bronchoalveolar lavage fluid and by areal lung inflammation, was significantly greater in mice inoculated with P . aeruginosa-laden agarose beads and Ad2/beta Gal-2 compared with controls . Mice that had received Pseudomonas-laden agarose beads and Ad2/beta Gal-2 had significantly fewer (P < 0.015) airway epithelial cells transduced (4.1 +/- 0.9%) compared with mice that received sterile agarose beads and Ad2/beta Gal-2 (9.4 +/- 1.4%) . These results indicate that the efficacy of adenovirus-mediated gene transfer is reduced in Pseudomonas-induced bronchopulmonary inflammation. J Bacteriol, 1998 Jun, 180(11), 2987 - 91 Role of the multidrug efflux systems of Pseudomonas aeruginosa in organic solvent tolerance; Li XZ et al.; Multidrug efflux pumps with a broad substrate specificity make a major contribution to intrinsic and acquired multiple antibiotic resistance in Pseudomonas aeruginosa . Using genetically defined efflux pump mutants, we investigated the involvement of the three known efflux systems, MexA-MexB-OprM, MexC-MexD-OprJ, and MexE-MexF-OprN, in organic solvent tolerance in this organism . Our results showed that all three systems are capable of providing some level of tolerance to organic solvents such as n-hexane and p-xylene . Expression of MexAB-OprM was correlated with the highest levels of tolerance, and indeed, this efflux system was a major contributor to the intrinsic solvent tolerance of P . aeruginosa . Intrinsic organic solvent tolerance was compromised by a protonophore, indicating that it is substantially energy dependent . These data suggest that the efflux of organic solvents is a factor in the tolerance of P . aeruginosa to these compounds and that the multidrug efflux systems of this organism can accommodate organic solvents, as well as antibiotics. J Bacteriol, 1998 Jun, 180(11), 2836 - 41 Effects of iron and temperature on expression of the Pseudomonas aeruginosa tolQRA genes: role of the ferric uptake regulator; Lafontaine ER et al.; The tolQRA genes have been recently identified in Pseudomonas aeruginosa PAO . In this study, we examined the effect of iron and temperature on tolQRA expression . A promoterless lacZ gene was introduced downstream of plasmid-encoded tolQ and tolA, and expression was monitored by measuring beta-galactosidase activity of cultures . Addition of 25 microM FeCl3 to the culture medium reduced tolQRA expression by 50 to 60% in PAO but by only 25% in the fur mutant PAO A4 . Northern hybridization analysis revealed that iron regulation occurs at the level of transcription and involves the P . aeruginosa ferric uptake regulator (Fur) . Primer extension analysis was used to identify the proposed transcriptional start site of tolA . Although a putative Fur box was identified 20 bp upstream of the proposed start site, purified Fur did not bind to the tolA or tolQR promoter regions in an in vitro gel retardation assay . Therefore, iron regulation of the tol genes appears to involve an intermediate regulatory gene . Expression of tolQR and tolA was optimal at 37 degrees C and was reduced by 40 to 50% when cultures were grown at either 42 or 25 degreesC . Growth in high-iron medium at 25 degrees C further reduced tolQR and tolA expression. Biochemistry, 1998 May 12, 37(19), 6689 - 96 Structure of the hexapeptide xenobiotic acetyltransferase from Pseudomonas aeruginosa; Beaman TW et al.; The crystal structure of the xenobiotic acetyltransferase from Pseudomonas aeruginosa PA103 (PaXAT) has been determined, as well as that of its complex with the substrate chloramphenicol and the cofactor analogue desulfo-coenzyme A . PaXAT is a member of the large hexapeptide acyltransferase family of enzymes that display tandem repeated copies of a six-residue hexapeptide repeat sequence motif encoding a left-handed parallel beta helix (L betaH) structural domain . The xenobiotic acetyltransferase class of hexapeptide acyltransferases is composed of microbial enzymes that utilize acetyl-CoA to acylate a variety of hydroxyl-bearing acceptors . The active site of trimeric PaXAT is a short tunnel into which chloramphenicol and the cofactor analogue desulfo-CoA project from opposite ends . This tunnel is formed by the flat parallel beta sheets of two separate L betaH domains and an extended 39-residue loop . His 79 of the extended loop forms hydrogen bonds from its imidazole NE2 atom to the 3-hydroxyl group of chloramphenicol and from its ND1 group to the peptide oxygen of Thr 86 . The interactions of this histidine residue are similar to those found in the structurally unrelated type III chloramphenicol acetyltransferase and suggest that His 79 of PaXAT may be similarly positioned and tautomerically stabilized to serve as a general base catalyst. Microbiology, 1998 May, 144 ( Pt 5), 1375 - 86 Regulation of the sulfate starvation response in Pseudomonas aeruginosa: role of cysteine biosynthetic intermediates; Hummerjohann J et al.; Pseudomonas aeruginosa PAO1 grew in defined synthetic medium with any of a broad variety of single sulfur sources, including sulfate, cysteine, thiocyanate, alkanesulfonates, organosulfate esters and methionine, but not with aromatic sulfonates, thiophenols or organothiocyanates or isothiocyanates . During growth with any of these compounds except sulfate, cysteine or thiocyanate, a set of 10 sulfate starvation-induced (SSI) proteins was strongly up-regulated, as observed by two-dimensional protein electrophoresis of total cell extracts . A comparable level of up-regulation was found for the hydrolytic enzyme arylsulfatase, which has previously been used as a marker enzyme for the sulfate starvation response . One of the SSI proteins was identified by N-terminal sequencing as a high-affinity periplasmic sulfate-binding protein, and another was related to thiol-specific antioxidants, but the N-terminal sequences of the other SSI proteins revealed no similarity to N-termini of proteins of known function, and they probably represent uncharacterized enzymes involved in sulfur scavenging when preferred sulfur sources are absent . To study the role that cysteine biosynthetic intermediates play in the synthesis of these proteins in vivo, we isolated mini-Tn5 transposon mutants of P . aeruginosa with insertions in the cysN and cysI genes, which encode subunits of ATP-sulfurylase and sulfite reductase, respectively . These two genes were cloned and sequenced . cysI showed high similarity to the cognate gene in Escherichia coli, whereas cysN encoded a 69.3 kDa protein with two domains corresponding to the E . coli CysN and CysC proteins . Sulfate no longer repressed synthesis of the SSI proteins in cysN mutants, but repression was restored by sulfite; in the cysI mutant, sulfate, sulfite and sulfide all led to repression of SSI protein synthesis . This suggests that there are at least two independent corepressors of the sulfate starvation response in this species. Mol Gen Mikrobiol Virusol, 1998, (2), 14 - 7 {Novel isoschizomers of restriction endonucleases from Pseudomonas aeruginosa strains}; Kravets AN et al.; Testing of Pseudomonas aeruginosa strains from the collection of the L . V . Gromashevsky Institute of Epidemiology and Infectious Diseases (Kiev) for specific endonucleases resulted in isolation of five class II restriction endonucleases, which were partially purified and their recognition targets were determined . Two of these endonucleases, Pae2kI and Pae18kI, are isoschizomers of Bg1II (5'-AGACTC-3') . Pae5kI and Pae14kI recognize the 5'-CCGC/GG-3' sequence and are therefore true isoschizomers of SacII . Hence, Pae17kI is an isoschizomer of PvuII and cleaves the DNA within the recognition sequence 5'-CAG/CTG-3' . Bg1II and PvuII are for the first time detected in Pseudomonas aeruginosa. Curr Microbiol, 1998 Jun, 36(6), 353 - 60 The characteristics of arylamine N-Acetyltransferase in Pseudomonas aeruginosa; Hsieh SE et al.; N-Acetyltransferase (NAT), responsible for bioactivation and detoxification of arylamines, has been demonstrated to be widely distributed in many organisms ranging from humans to microorganisms . Using high performance liquid chromatography (HPLC) to analyze NAT activity in bacteria, the authors found that Pseudomonas aeruginosa exhibited high NAT activity with 2-aminofluorene (2-AF) as substrate . Characteristics of this bacterial NAT were further investigated . The N-acetylation catalyzed by this enzyme is an acetyl coenzyme A (AcCoA)-dependent reaction . As the concentration of AcCoA in the reaction mixture was increased, the apparent K(m) and Vmax for 2-AF increased . The K(m) and Vmax were 0.504 +/- 0.056 mM and 31.92 +/- 3.23 nmol/min/mg protein, respectively, for the acetylation of 2-AF with 0.5 mM AcCoA . The optimum pH for the enzyme activity was estimated to be around 8.5 . It was active at a temperature range from 5 degrees C to 55 degrees C, with maximum activity at 37 degrees C . The enzyme activity was inhibited by divalent metal ions including Cu++, Fe++, Zn++, Ca++, Co++, Mn++, and Mg++, suggesting that a sulfhydryl group is involved in the N-acetylation activity . The three chemical modification agents, iodoacetamide, phenylglyoxal, and diethylpyrocarbonate, all exhibited a dose-, time-, and temperature-dependent inhibition effect . Preincubation of the NAT with AcCoA provided significant protection against the inhibition of iodoacetamide and diethylpyrocarbonate, but only partial protection against the inhibition of phenylglyoxal . These results indicate that cysteine, histidine, and arginine residues are essential for this bacterial enzyme activity, and the first two are likely to reside on the AcCoA binding site, but arginine residue may be located only near the AcCoA binding site . Our data demonstrate that P . aeruginosa possesses highly active N-acetyltransferase which shares a similar catalytic mechanism as that of higher organisms . These findings are very helpful for further investigating the role of arylamine NAT in this bacterial species. Acta Virol, 1997 Oct, 41(5), 293 - 6 Transduction of antibiotic resistance including imipenem resistance by wild type phages from nosocomial strains of Pseudomonas aeruginosa; Blahova J et al.; In this report we describe transduction of antibiotic resistance determinants by three wild type bacteriophages isolated from three Pseudomonas aeruginosa strains . The strains showed evident plaques of a lysis caused by a bacteriophage . The strains were identified as lysogenic among 31 imipenem (IMP)-resistant P . aeruginosa strains isolated at the National Institute of Oncology in Bratislava . The carbenicillin (CAR) resistance determinant was transduced by all the three phages to four P . aeruginosa recipients-PAO-1670, ML-M-88, ML-1292 and ML-1008 . The gentamicin (GEN) resistance was transduced to ML-1008 only . The kanamycin (KAN) resistance was transduced in the following systems (combinations): "phage AP-37 to M-88", "phage AP-38 to PAO-1670, ML-1292 and M-88", and "phage AP-40 to M-88" . The IMP resistance determinant was transduced by all the three phages to P . aeruginosa recipient strains . All transductant colonies were tested for the presence of directly not selected but co-transduced resistance determinants . Whereas transductants selected on media with IMP were resistant to five antibiotics (IMP, CAR, streptomycin (STR), KAN and GEN), transductants selected on CAR, KAN, STR, or GEN were resistant to a block of four of these antibiotics but not to IMP. Vaccine, 1998 Jan, 16(1), 76 - 82 Mitogenic synthetic polynucleotides suppress the antibody response to a bacterial polysaccharide; Threadgill DS et al.; Unmethylated bacterial DNA containing a high frequency of the CpG motif, is mitogenic and induces T-cell independent, murine B-cell proliferation . These stimulatory effects are also induced by synthetic oligonucleotides that contain one or more unmethylated CpG dinucleotides (CpG oligo) . Such mitogenicity is not seen with highly methylated vertebrate DNA, which has a lower prevalence of the CpG motif than bacterial DNA . Due to their stimulatory effects, CpG oligo have been proposed for use as vaccine adjuvants . In order to determine if a synthetic CpG oligo that was stimulatory for B-cell proliferation could augment the murine antibody response to protective bacterial polysaccharide epitopes (Pseudomonas aeruginosa LPS-O polysaccharide side chain; high-molecular-weight polysaccharide or high-MW PS), BALB/c mice were injected with mitogenic doses of CpG oligo simultaneously with high-MW PS, and antibody titers were measured by ELISA weekly for 4 weeks . Controls received PBS, a nonstimulatory control oligo plus PS, CpG alone, or PS alone . Despite evidence of B-cell mitogenicity and an increase in total IgM in CpG oligo-treated mice, CpG oligo treatment plus PS significantly decreased the high-MW PS antibody response compared to PS alone . The blunting of the anti-PS antibody response could be eliminated by vaccinating the animals with PS prior to CpG oligo . We conclude that despite in vitro and in vivo evidence of B-cell proliferation, this CpG oligo reduces PS-specific antibody responses in an animal model when given simultaneously with a bacterial polysaccharide . Based on results in this model, oligonucleotides containing stimulatory unmethylated CpG dinucleotides may not be useful adjuvants when given simultaneously with bacterial PS vaccines. Can J Microbiol, 1998 Mar, 44(3), 307 - 11 Interaction between the pili of Pseudomonas aeruginosa PAK and its carbohydrate receptor beta-D-GalNAc(1-->4)beta-D-Gal analogs; Schweizer F et al.; Pseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surface receptors . Previously, it has been shown that the pilus adhesin of P . aeruginosa PAK binds to the ganglioside asialo-GM1 . In particular, it was found that the carbohydrate sequence beta-D-GalNAc(1-->4)beta-D-Gal is the minimal carbohydrate receptor sequence of asialo-GM1 . To study the binding specificity of P . aeruginosa, O-modified and N-modified sugar analogs, where each hydroxyl group was substituted either by O-methyl or O-propyl and the acetamido group was changed to a propionamido group, were synthesized . The sugar analogs were evaluated as inhibitors in a competitive solid phase binding assay . The results demonstrate that the pili of P . aeruginosa PAK accepts a variety of sugar analogs possessing the sequence beta-D-GalNAc(1-->4)beta-D-Gal . Most sugar analogs bind with a similar order of magnitude (50% inhibitory concentration (IC50) = 60-130 microM) except for the 2-O-propyl derivative 7 (IC50 = 8 +/- 4 microM) compared with an IC50 of 79 +/- 18 microM for the native compound . The significant increase in binding affinity of 2-O-propyl derivative 7 suggests that improved inhibitors of adhesion may be prepared by introducing a hydrophobic side chain at the 2-position of galactose. Cornea, 1998 May, 17(3), 293 - 300 Efficacy of ophthalmic solutions to detach adhering Pseudomonas aeruginosa from contact lenses; Landa AS et al.; PURPOSE: To compare the efficacies of two all-in-one contact lens (CL) cleaning solutions and a detergent mixture on the detachment of a pathogenic bacterium adhering to two types of contact lenses in the absence and presence of a tear film . METHODS: Bacterial-detachment studies were carried out in a parallel-plate flow chamber . Rigid gas permeable (RGP) CLs with and without a tear film were fixed on the bottom plate of the flow chamber . After adhesion of Pseudomonas aeruginosa no . 3, bacterial detachment was stimulated by perfusing the system either with an all-in-one CL-cleaning solution, for soft contact lenses (SCL solution) and for rigid lenses (RCL solution), or with a detergent mixture of 0.25% (wt/vol) sodium lauryl sulfate (SLS) and 0.2% sodium methyl cocoyl taurate (Tauranol) . In addition, the all-in-one RCL-cleaning solution supplemented with 0.025% (wt/vol) SLS and 0.02% (wt/vol) Tauranol was evaluated . A surface physical-chemical analysis of the lenses before and after application of the solutions was done to determine whether remnants of the ophthalmic solutions or detergents could be found adsorbed to the CL surfaces . RESULTS: Both all-in-one CL-cleaning solutions stimulated minor bacterial detachment from CL surfaces with or without a tear film . The SLS/Tauranol detergent mixture, however, removed < or = 95% of the adhering P . aeruginosa cells, whereas the RCL-cleaning solution supplemented with detergents also stimulated significant detachment . Surface physical-chemical analysis clearly demonstrated the presence of a tear film on the CL surfaces, but remnants neither of the ophthalmic solutions nor of the detergents could be found . CONCLUSION: Ophthalmic solutions are not effective in stimulating detachment of adhering bacteria from CL surfaces . Supplementing of an all-in-one CL-cleaning solution with only small amounts of detergents yielded a solution much more effective in stimulating bacterial detachment while leaving no detectable remnants of the ophthalmic solution or of the detergents on the CL surfaces. Infect Immun, 1998 Jun, 66(6), 2607 - 13 Modification of Ras in eukaryotic cells by Pseudomonas aeruginosa exoenzyme S; McGuffie EM et al.; Genetic and functional data suggest that Pseudomonas aeruginosa exoenzyme S (ExoS), an ADP-ribosyltransferase, is translocated into eukaryotic cells by a bacterial type III secretory mechanism activated by contact between bacteria and host cells . Although purified ExoS is not toxic to eukaryotic cells, ExoS-producing bacteria cause reduced proliferation and viability, possibly mediated by bacterially translocated ExoS . To investigate the activity of translocated ExoS, we examined in vivo modification of Ras, a preferred in vitro substrate . The ExoS-producing strain P . aeruginosa 388 and an isogenic mutant strain, 388DeltaexoS, which fails to produce ExoS, were cocultured with HT29 colon carcinoma cells . Ras was found to be ADP-ribosylated during coculture with 388 but not with 388DeltaexoS, and Ras modification by 388 corresponded with reduction in HT29 cell DNA synthesis . Active translocation by bacteria was found to be required, since exogenous ExoS, alone or in the presence of 388DeltaexoS, was unable to modify intracellular Ras . Other ExoS-producing strains caused modification of Ras, indicating that this is not a strain-specific event . ADP-ribosylation of Rap1, an additional Ras family substrate for ExoS in vitro, was not detectable in vivo under conditions sufficient for Ras modification, suggesting possible ExoS substrate preference among Ras-related proteins . These results confirm that intracellular Ras is modified by bacterially translocated ExoS and that the inhibition of target cell proliferation correlates with the efficiency of Ras modification. Infect Immun, 1998 Jun, 66(6), 2521 - 8 Pseudomonas aeruginosa lasR transcription correlates with the transcription of lasA, lasB, and toxA in chronic lung infections associated with cystic fibrosis; Storey DG et al.; The role of Pseudomonas aeruginosa quorum-sensing systems in the lung infections associated with cystic fibrosis (CF) has not been examined . The purpose of this study was to determine if genes regulated by the LasR-LasI quorum-sensing system were coordinately regulated by the P . aeruginosa populations during the lung infections associated with CF . We also wanted to ascertain if there was a relationship between the expression of lasR, a transcriptional regulator, and some P . aeruginosa virulence factors during these infections . We extracted RNAs from the bacterial populations of 131 sputa taken from 23 CF patients . These RNAs were blotted and hybridized with probes to P . aeruginosa lasA, lasB, and toxA . The hybridization signals from each probe were ranked, and the rankings were analyzed by a Spearman rank correlation to determine if there was an association between the population transcript accumulations for the three genes . The correlations between the transcript accumulation patterns of pairs of the genes suggested that lasA, lasB, and toxA might be coordinately regulated during CF lung infections . To determine if this coordinate regulation might be due to regulation by LasR, we probed RNAs, extracted from 84 sputa, with the lasR, lasA, lasB, toxA, and algD probes . Statistical analysis indicated that lasR transcript accumulation correlated to lasA, lasB, toxA, and algD transcript accumulations . These results indicated that lasR may at least partially regulate or be coordinately regulated with lasA, lasB, toxA, and algD during the lung infections associated with CF . These results also suggested that the LasR-LasI quorum-sensing system may control the expression of at least some virulence factors in the lungs of patients with CF. Infect Immun, 1998 Jun, 66(6), 2447 - 52 Lipopolysaccharide-related stimuli induce expression of the secretory leukocyte protease inhibitor, a macrophage-derived lipopolysaccharide inhibitor; Jin F et al.; Mouse secretory leukocyte protease inhibitor (SLPI) was recently characterized as a lipopolysaccharide (LPS)-induced product of macrophages that antagonizes their LPS-induced activation of NF-kappaB and production of NO and tumor necrosis factor (TNF) (F . Y . Jin, C . Nathan, D . Radzioch, and A . Ding, Cell 88:417-426, 1997) . To better understand the role of SLPI in innate immune and inflammatory responses, we examined the kinetics of SLPI expression in response to LPS, LPS-induced cytokines, and LPS-mimetic compounds . SLPI mRNA was detectable in macrophages by Northern blot analysis within 30 min of exposure to LPS but levels peaked only at 24 to 36 h and remained elevated at 72 h . Despite the slowly mounting and prolonged response, early expression of SLPI mRNA was cycloheximide resistant . Two LPS-induced proteins-interleukin-10 (IL-10) and IL-6-also induced SLPI, while TNF and IL-1beta did not . The slow attainment of maximal induction of SLPI by LPS in vitro was mimicked by infection with Pseudomonas aeruginosa in vivo, where SLPI expression in the lung peaked at 3 days . Two LPS-mimetic molecules-taxol from yew bark and lipoteichoic acid (LTA) from gram-positive bacterial cell walls-also induced SLPI . Transfection of macrophages with SLPI inhibited their LTA-induced NO production . An anti-inflammatory role for macrophage-derived SLPI seems likely based on SLPI's slowly mounting production in response to constituents of gram-negative and gram-positive bacteria, its induction both as a direct response to LPS and as a response to anti-inflammatory cytokines induced by LPS, and its ability to suppress the production of proinflammatory products by macrophages stimulated with constituents of both gram-positive and gram-negative bacteria. Glycobiology, 1998 Jan, 8(1), 7 - 16 Studies on the binding site of the galactose-specific agglutinin PA-IL from Pseudomonas aeruginosa; Chen CP et al.; The binding properties of Pseudomonas aeruginosa agglutinin-I (PA-IL) with glycoproteins (gps) and polysaccharides were studied by both the biotin/avidin-mediated microtiter plate lectin-binding assay and the inhibition of agglutinin-glycan interaction with sugar ligands . Among 36 glycans tested for binding, PA-IL reacted best with two glycoproteins containing Galalpha1-->4Gal determinants and a human blood group ABO precursor equivalent gp, but this lectin reacted weakly or not at all with A and H active gps or sialylated gps . Among the mammalian disaccharides tested by the inhibition assay, the human blood group Pkactive Galalpha1-->4Gal, was the best . It was 7.4-fold less active than melibiose (Galalpha1-->6Glc) . PA-IL has a preference for the alpha-anomer in decreasing order as follows: Galalpha1-->6 >Galalpha1-->4 >Galalpha1-->3 . Of the monosaccharides studied, the phenylbeta derivatives of Gal were much better inhibitors than the methylbeta derivative, while only an insignificant difference was found between the Galalpha anomer of methyl- and p -NO2-phenyl derivatives . From these results, it can be concluded that the combining size of the agglutinin is as large as a disaccharide of the alpha-anomer of Gal at nonreducing end and most complementary to Galalpha1-->6Glc . As for the combining site of PA-IL toward the beta-anomer, the size is assumed to be less than that of Gal; carbon-6 in the pyranose form is essential, and hydrophobic interaction is important for binding. J Clin Microbiol, 1998 Apr, 36(4), 897 - 901 Emergence of multidrug resistance in ubiquitous and dominant Pseudomonas aeruginosa serogroup O:11 . The Greek Pseudomonas Aeruginosa Study Group; Tassios PT et al.; The serotypes of 88 nonreplicate nosocomial Pseudomonas aeruginosa isolates from 11 Greek hospitals were studied in relation to their antibiotic susceptibilities . Rates of resistance to beta-lactams, aminoglycosides, and quinolones ranged from 31 to 65%, except for those to ceftazidime (15%) and imipenem (21%) . Four serotypes were dominant: O:12 (25% of isolates), O:1 (17%), O:11 (16%), and O:6 (10%) . Multidrug resistance rates in the major serogroups O:12 (91%) and O:11 (79%) were higher than those in serogroups O:1 (40%) and O:6 (43%) . Further typing with respect to pulsed-field gel electrophoresis patterns following XbaI digestion of genomic DNA discriminated the isolates into 74 types . Pulsed-field gel electrophoresis revealed that the ubiquitous O:12 group was genetically homogeneous, since 95% of strains belonged to two clusters of genotypic similarity, while the O:11 strains, present in 8 of the 11 hospitals, were distributed among five such clusters . Therefore, apart from the already reported O:12 multidrug-resistant European clone, an O:11 population, characterized by a serotype known to be dominant in the environment and the hospital in several parts of the world, but previously not associated with multidrug resistance to antibiotics, has progressed to a multidrug-resistant state. Chest, 1998 May, 113(5), 1225 - 9 Pulmonary complications of HIV infection: autopsy findings; Afessa B et al.; STUDY OBJECTIVES: To describe the pulmonary complications in patients with HIV infection, and the changes in the incidence of these complications over a 12-year period . DESIGN: Retrospective review of autopsy records . SETTING: Two university-affiliated medical centers . PATIENTS: We studied autopsy findings from 233 patients with HIV infection who died between 1985 and 1996 . Demographic data, risk factors for HIV infection, and the lengths of hospital stay were obtained . The histologic and microbiological findings of the respiratory system, and the extrapulmonary organ involvement by Kaposi's sarcoma (KS), Pneumocystis carinii, Mycobacterium tuberculosis, and Mycobacterium avium complex were reviewed . RESULTS: Ninety-two percent of the patients were black and 75% were male . The two most common identified risk factors for HIV infection were homosexuality (34%) and injection drug use (27%) . Bacterial pneumonia was the most frequent pulmonary complication (42%) . The two most common causes of bacterial pneumonia were Pseudomonas aeruginosa and Staphylococcus aureus . P carinii pneumonia (PCP) was found in 24%, with extrapulmonary involvement in 13% . Pulmonary mycobacterial infections were seen in 33%, with multiple extrapulmonary involvement . The most common site affected by KS was the lung . Of all pulmonary complications, only the incidence of PCP decreased over the 12-year period . CONCLUSIONS: Recognizing the high incidence rate of bacterial pneumonia, the high frequency of pulmonary KS and the not uncommon occurrence of extrapulmonary P carinii infection in patients with HIV helps in improving their care. FEMS Microbiol Lett, 1998 May 1, 162(1), 123 - 6 Constitutive choline transport in Pseudomonas aeruginosa; Lucchesi GI et al.; Pseudomonas aeruginosa has a choline uptake system which is expressed in bacteria grown in the presence of succinate and ammonium chloride as the carbon and nitrogen source, respectively . This system obeys Michaelis-Menten kinetics with an apparent Km value of 53 microM; its activity is not inhibited by high osmolarities in the medium but is partially inhibited by choline metabolites such as betaine and dimethylglycine. FEMS Microbiol Lett, 1998 May 1, 162(1), 31 - 7 Involvement of the RpoN protein in the transcription of the oprE gene in Pseudomonas aeruginosa; Yamano Y et al.; OprE is a channel-forming outer membrane protein of Pseudomonas aeruginosa, the expression of which is induced under anaerobic conditions . We constructed various mutants and observed the effects on oprE expression . Deficiency in RpoN, an alternative sigma factor for RNA polymerase, abolished oprE expression under aerobic conditions, but did not affect the expression under anaerobic conditions . One mutation on the putative RpoN recognition site also caused reduction of oprE expression . The region 500 nucleotides upstream of the mRNA start site was required for optimal oprE transcription, which contains an AT-rich region including a putative integration host factor binding site . These results indicate that OprE production is directly or indirectly controlled by RpoN but also require some other regulatory proteins bound to the upstream region. Mol Microbiol, 1998 Apr, 28(1), 193 - 203 In vitro biosynthesis of the Pseudomonas aeruginosa quorum-sensing signal molecule N-butanoyl-L-homoserine lactone; Jiang Y et al.; In Pseudomonas aeruginosa, synthesis of the quorum-sensing signal molecules N-butanoyl-L-homoserine lactone (BHL) and N-hexanoyl-L-homoserine lactone (HHL) requires the Luxl homologue Rhll(Vsml) . By using thin-layer chromatography in conjunction with high-performance liquid chromatography (HPLC) and mass spectrometry, we show that purified Rhll can catalyse the biosynthesis of BHL and HHL using either S-adenosylmethionine (SAM) or homoserine lactone (HSL) but not homoserine as the source of the homoserine lactone moiety . As we were unable to detect homoserine lactone in cytoplasmic extracts of Escherichia coli, we conclude that SAM is the natural substrate for Rhll-directed N-acylhomoserine lactone (AHL) biosynthesis . The N-acyl chain of BHL and HHL can be supplied by the appropriately charged coenzyme A derivative (either n-butanoyl-CoA or n-hexanoyl-CoA) . The specificity of Rhll for charged CoA derivatives is demonstrated as Rhll was unable to generate AHLs detectable in our bioassays from acetyl-CoA, malonyl-CoA, n-octanoyl-CoA, n-decanoyl-CoA, DL-beta-hydroxybutanoyl-CoA or crotonoyl-CoA . Rhll was also unable to use N-acetyl-S-3-oxobutanoylcysteamine, a chemical mimic for 3-oxobutanoyl-CoA . Furthermore, the Rhll-catalysed synthesis of BHL and HHL was most efficiently driven when NADPH was included in the reaction mixture. Antimicrob Agents Chemother, 1998 May, 42(5), 1015 - 21 Biological characterization of endotoxins released from antibiotic-treated Pseudomonas aeruginosa and Escherichia coli; Kirikae T et al.; The supernatants taken from Pseudomonas aeruginosa and Escherichia coli cultures in human sera or chemically defined M9 medium in the presence of ceftazidime (CAZ) contained high levels of endotoxin, while those taken from the same cultures in the presence of imipenem (IPM) yielded a very low level of endotoxin . The biological activities of endotoxin in the supernatants were compared with those of phenol water-extracted lipopolysaccharide (LPS) . The endotoxin released from the organisms as a result of CAZ treatment (CAZ-released endotoxin) contained a large amount of protein . The protein, however, lacked endotoxic activity, since the endotoxin did not show any in vivo toxic effects in LPS-hyporesponsive C3H/HeJ mice sensitized with D-(+)-galactosamine (GalN) or any activation of C3H/HeJ mouse macrophages in vitro . The activities of CAZ- and IPM-released endotoxin (as assessed by a chromogenic Limulus test) were fundamentally the same as those of P . aeruginosa LPS, since their regression lines were parallel . The CAZ-released endotoxin was similar to purified LPS with respect to the following biological activities in LPS-responsive C3H/HeN mice and LPS-hyporesponsive C3H/HeJ mice: lethal toxicity in GalN-sensitized mice, in vitro induction of tumor necrosis factor- and NO production by macrophages, and mitogen-activated protein kinase activation in macrophages . The macrophage activation by CAZ-released endotoxin as well as LPS was mainly dependent on the presence of serum factor and CD14 antigen . Polymyxin B blocked the activity . These findings indicate that the endotoxic activity of CAZ-released endotoxin is due primarily to LPS (lipid A). Antimicrob Agents Chemother, 1998 May, 42(5), 1005 - 11 Characteristics and dynamics of bacterial populations during postantibiotic effect determined by flow cytometry; Gottfredsson M et al.; Changes in bacterial ultrastructure after antibiotic exposure and during the postantibiotic effect (PAE) have been demonstrated by electron microscopy (EM) . However, EM is qualitative and subject to individual interpretation . In contrast, flow cytometry gives qualitative and quantitative information . The sizes and nucleic acid contents of Escherichia coli and Pseudomonas aeruginosa were studied during antimicrobial exposure as well as during the PAE period by staining the organisms with propidium iodide and analyzing them with flow cytometry and fluorescence microscopy . The effects of ampicillin, ceftriaxone, ciprofloxacin, gentamicin, and rifampin were studied for E . coli, whereas for P . aeruginosa imipenem and ciprofloxacin were investigated . After exposure of E . coli to ampicillin, ceftriaxone, and ciprofloxacin, filamentous organisms were observed by fluorescence microscopy . These changes in morphology were reflected by increased forward light scatter (FSC) and nucleic acid content as measured by flow cytometry . For the beta-lactams the extent of filamentation increased in a dose-dependent manner after drug removal, resulting in formation of distinct subpopulations of bacteria . These changes peaked at 20 to 35 min, and bacteria returned to normal after 90 min after drug removal . In contrast, the subpopulations induced by ciprofloxacin did not return to normal until > 180 min after the end of the classically defined PAE . Rifampin resulted in formation of small organisms with low FSC, whereas no distinctive characteristics were noted after gentamicin exposure . For P . aeruginosa an identifiable subpopulation of large globoid cells and increased nucleic acid content was detected after exposure to imipenem . These changes persisted past the PAE, as defined by viability counting . Swollen organisms with increased FSC were detected after ciprofloxacin exposure, even persisting during bacterial growth . In summary, for beta-lactam antibiotics and ciprofloxacin, the PAE is characterized by dynamic formation of enlarged cell populations of increased nucleic acid content, whereas rifampin induces a decrease in size and nucleic acid content in the organisms . Flow cytometry is an ideal method for future studies of bacterial phenotypic characteristics during the PAE. Mikrobiologiia, 1998 Jan-Feb, 67(1), 102 - 5 {Survival of dissociants of a hydrocarbon-oxidizing Pseudomonas aeruginosa strain during storage}; Mil'ko ES; The survival of R-, S-, and M-dissociants of the hydrocarbon-oxidizing strain Pseudomonas aeruginosa K-2 was compared after the storage of the cultures by four simple methods for 18 months . The best method was storage on slanted synthetic medium with hexadecane under mineral oil; under these conditions, the amount of viable cells of all dissociants showed the smallest decrease, and the composition of the stored populations underwent the least changes . During storage by other methods, the highest survival rate was demonstrated by R-cells, and M-cells were the most sensitive; this difference resulted in changes in the composition of stored populations . In stored populations of S-cells, which are the most active hydrocarbon oxidizers, the relative content of poorly active but more stable R-cells increased, which could result in a decrease in the hydrocarbon-degrading ability of the culture. Electrophoresis, 1998 Apr, 19(4), 509 - 14 Differential genome analysis of bacteria by genomic subtractive hybridization and pulsed field gel electrophoresis; Schmidt KD et al.; A comprehensive analysis of the differences between the genomes of two closely related bacterial strains should give insight into the molecular basis of their individual phenotypic and genotypic characteristics . Here we present an integrative approach including two different strategies for the thorough investigation of genomic divergence . We have combined two techniques including genomic subtractive hybridization and comparative genome mapping by pulsed field gel electrophoresis (PFGE) techniques . The subtractive method for which a protocol is given herein results in the production of a library of specific DNA sequence tags present only in one strain, while the construction of macrorestriction maps of the bacterial chromosomes yields data about the overall genome organization and the arrangement and distance of gene loci . Comparison of the physical and genetic maps and determination of the map positions of the strain-specific DNA sequences reveals gross chromosomal modifications, insertions or deletions of additional genetic material, and transpositional events . The further investigation of the strain-specific regions yields information about the nature and origin of the acquired DNA and their influence on the evolution of the individual bacterial genome . The two methods were applied to differential genome analysis of clonal divergence in Pseudomonas aeruginosa choosing two clone C isolates from diverse habitats. Electrophoresis, 1998 Apr, 19(4), 495 - 9 Smith/Birnstiel mapping of genome rearrangements in Pseudomonas aeruginosa; Heuer T et al.; A novel application of the Smith/Birnstiel technique is presented for the analysis of intraspecies genomic diversity in small genomes . Rare-cutter total/N frequent-cutter partial restriction digestions are separated in N separate lanes by pulsed-field gel electrophoresis, blotted and hybridized with rare-cutter fragment ends as probes . The evaluation of the autoradiogram results in high-resolution restriction maps of 10-200 kbp regions . The technique was applied to the analysis of genome rearrangements in Pseudomonas aeruginosa strains . Comparison of the region encoding the tryptophan biosynthesis genes in the PAO and the IATS serotype 5 strains revealed that shared sequence characterized by almost identical restriction fingerprints was interrupted in the serotype 5 strain by small islands displaying strain-specific restriction site signatures . A multistep rearrangement in a hypervariable chromosome region downstream of the phn locus was detected in serial airway isolates from a patient with cystic fibrosis. Electrophoresis, 1998 Apr, 19(4), 486 - 94 Cloning of prokaryotic genomes in yeast artificial chromosomes: application to the population genetics of Pseudomonas aeruginosa; Heuer T et al.; Yeast artificial chromosomes (YACs) can accommodate large inserts and hence should be attractive tools for intra- and interspecies comparisons of bacterial genomes . YAC libraries were constructed from size-selected partial digests of human and Pseudomonas aeruginosa PAO DNA and SpeI-restricted PAO DNA . Whereas YACs from human DNA had an average size of 350 kilobase pairs (kbp), a P . aeruginosa sequence larger than 120 kbp was absent or truncated in the eukaryotic host . Coligation occurred for YACs smaller than 40 kbp, but stable YACs with 40-120 kbp large inserts of P . aeruginosa DNA were obtained in high yield . SpeI-restricted chromosomes from 97 P . aeruginosa strains representing 47 genotypes were hybridized with stable YACs from three equidistant regions of the PAO genome . The low complexity of hybridizing bands demonstrated that the analyzed 100 kbp sequence contigs were stably maintained in most P . aeruginosa isolates from both disease and environmental habitats. Proc Natl Acad Sci U S A, 1998 May 12, 95(10), 5718 - 23 Activation of NF-kappaB via a Src-dependent Ras-MAPK-pp90rsk pathway is required for Pseudomonas aeruginosa-induced mucin overproduction in epithelial cells; Li JD et al.; Cystic fibrosis (CF) is an autosomal recessive disorder, the most common lethal genetic disease in Caucasians . Respiratory disease is the major cause of morbidity and mortality . Indeed, 95% of CF patients die of respiratory failure . Pseudomonas aeruginosa, an opportunistic pathogen, chronically infects the lungs of over 85% of CF patients . It is ineradicable by antibiotics and responsible for airway mucus overproduction that contributes to airway obstruction and death . The molecular mechanisms underlying this pathology are unknown . Here we show that P . aeruginosa activates a c-Src-Ras-MEK1/2-MAPK-pp90rsk signaling pathway that leads to activation of nuclear factor NF-kappaB (p65/p50) . Activated NF-kappaB binds to a kappaB site in the 5'-flanking region of the MUC2 gene and activates MUC2 mucin transcription . These studies bring new insight into bacterial-epithelial interactions and more specifically into the molecular pathogenesis of cystic fibrosis . Understanding these signaling and gene regulatory mechanisms opens up new therapeutic targets for cystic fibrosis. Anesth Analg, 1998 May, 86(5), 1075 - 8 Halogenated anesthetics inhibit Pseudomonas aeruginosa growth in culture conditions reproducing the alveolar environment; Molliex S et al.; We assessed the effects of halogenated anesthetics on Pseudomonas aeruginosa growth in a liquid nutrient broth . Sterile Petri dishes (3.5-cm diameter) were filled with a 1-mL suspension of a Pseudomonas aeruginosa strain and incubated at 37 degrees C . Exposure of bacterial plates to halothane, isoflurane, and enflurane administered at 1 and 2 minimum alveolar anesthetic concentration (MAC) were studied for different exposure times (1, 2, 3, and 4 h) using an airtight chamber . For each time, a control point was obtained . Serial dilutions and agar plates were made, and developed colonies were counted . A significant decrease in bacterial growth was observed from the second hour of exposure to every halogenated anesthetic . For long periods of exposure (3 and 4 h), bacterial growth was significantly reduced in the plates exposed to 2 MAC compared with 1 MAC . The maximal inhibition was observed after a 4-h exposure at 2 MAC and reached 60%, 49%, and 42% for halothane, isoflurane, and enflurane, respectively . We conclude that a decrease in Pseudomonas aeruginosa growth is observed after exposure to halogenated anesthetics, but whether this inhibition is clinically important remains to be demonstrated . Implications: Bacterial pneumonia is a major source of morbidity after general anesthesia . We measured the effects of volatile anesthetics on the growth of Pseudomonas aeruginosa, one of the pathogens most often isolated in hospital-acquired pneumonia . The experiments were performed in vitro in culture conditions reproducing those observed in the alveolar space . Volatile anesthetics inhibited the growth of these bacteria, but the clinical significance of this fact remains to be determined. J Laryngol Otol, 1998 Jan, 112(1), 98 - 102 Necrotizing external otitis caused by Aspergillus fumigatus: computed tomography and high resolution magnetic resonance imaging in an AIDS patient; Munoz A et al.; Most necrotizing (malignant) external otitis (NEO) occurs in diabetic patients and is commonly caused by Pseudomonas aeruginosa . We report an acquired immunodeficiency syndrome (AIDS) patient with NEO caused by Aspergillus fumigatus in which computed tomography (CT) showed destructive petrous bone involvement and magnetic resonance imaging (MRI) of the ear discovered extensive soft tissue and facial nerve involvement . Dedicated MRI studies of the ear in this type of pathology provide new insights relating to nerve dysfunction, that cannot be obtained with CT. Microbiology, 1998 Apr, 144 ( Pt 4), 1021 - 31 Carbapenems as inhibitors of OXA-13, a novel, integron-encoded beta-lactamase in Pseudomonas aeruginosa; Mugnier P et al.; A clinical Pseudomonas aeruginosa strain, PAe391, was found to be resistant to a number of antibiotics including ticarcillin, piperacillin, cefsulodin and amikacin, and a disk diffusion assay showed evidence of pronounced synergy between imipenem and various beta-lactam antibiotics . Cloning and nucleotide sequence analysis revealed the dicistronic arrangement of an aac(6')-Ib variant and a novel blaOXA-type gene between the intI and qacE delta 1 genes typical of integrons, in PAe391, this integron was apparently chromosome-borne . The beta-lactamase, named OXA-13, displayed nine amino acid changes with respect to OXA-10:I in position 10 of OXA-10 to T (I10T), G20S, D55N, N73S, T107S, Y174F, E229G, S245N and E259A, OXA-13 (pIapp = 8.0) showed poor catalytic activity against penicillins as well as cephalosporins, but was efficient in hydrolysing some penicillinase-resistant beta-lactams, such as cefotaxime and aztreonam . It was efficiently inhibited by imipenem (KIapp = 11 nM), and formed a stable complex . While the KIapp value of meropenem was similar (16 nM), the corresponding complex was less stable. J Antimicrob Chemother, 1998 Mar, 41(3), 407 - 9 Treatment of Pseudomonas aeruginosa lung infection in cystic fibrosis with high or conventional doses of ceftazidime; De Boeck K et al.; In cystic fibrosis patients with Pseudomonas aeruginosa colonization and increasing pulmonary infection, ceftazidime 150 mg/kg/day was compared with 320 mg/kg/day . Changes in clinical findings, laboratory tests, pulmonary function and chest radiographs were evaluated after 14 days of treatment in hospital . Both treatments were associated with a significant improvement, but the higher dose did not offer an additional benefit . An increase in alanine aminotransferase (ALT) occurred after both treatments; with a significantly greater increase after the high-dose therapy (mean increase +/- S.E.M . 8% +/- 2% vs 2% +/- 1 %; P < 0.01) . All but one of the ALT values after treatment were within normal limits. J Antimicrob Chemother, 1998 Mar, 41(3), 403 - 5 Comparative therapeutic efficacy of clinafloxacin in a Pseudomonas aeruginosa mouse renal abscess model; Shapiro MA et al.; A deep-seated Pseudomonas aeruginosa mouse kidney abscess model was used to compare the therapeutic efficacy of clinafloxacin, a fluoroquinolone in clinical trials, with that of clinically relevant standard drugs . Following 50 mg/kg oral doses, twice daily for five consecutive days, clinafloxacin produced a 4 log decrease in mean bacterial count, the greatest decrease of all drugs tested . The same dosage regimen resulted in complete bacterial eradication in 88% of the kidneys . No other compound produced total bacterial clearance in 50% of the kidneys at the highest dose tested. Int J Pediatr Otorhinolaryngol, 1998 Mar 1, 43(2), 141 - 5 Neonatal suppurative submandibular sialadenitis: a rare clinical entity; Ungkanont K et al.; Submandibular sialadenitis is very rare in the neonatal age group . Only four cases of isolated submandibular gland infection without the involvement of the parotid gland have been reported in the literature up to the present time . We present a case of a premature newborn infant who developed fever and right submandibular swelling when he was 8 days old . Pus was found extruding from Wharton's duct . The pus culture yielded Pseudomonas aeruginosa . The patient was treated conservatively with adequate hydration and intravenous ceftazidime for 14 days and the infection resolved . The possible predisposing factors were dehydration and prematurity . CT scan revealed no calculi and no anatomic derangement . Two months follow-up showed no evidence of recurrence . Previous literature is reviewed and discussed. J Small Anim Pract, 1998 Apr, 39(4), 165 - 8 Use of ticarcillin in the management of canine otitis externa complicated by Pseudomonas aeruginosa; Nuttall TJ; Twelve dogs were referred with purulent and proliferative otitis externa . Prior treatment included fluoroquinolones, glucocorticoids and polyvalent ear drops over seven days to five months . In all cases the vertical and horizontal ear canals were inflamed and thickened, with ruptured tympanic membranes in four cases . No abnormalities were seen on radiography of the osseous bullae . Numerous rod bacilli and degenerate neutrophils were seen on cytology . Pseudomonas aeruginosa resistant to fluoroquinolones and gentamicin was cultured in all cases . Treatment was initiated with 1 to 2 mg/kg prednisolone per os once daily, and a cleansing and drying ear cleaner followed by topical administration of injectable ticarcillin solution four times daily . Cases with ruptured tympanae also received 15 to 25 mg/kg ticarcillin three times daily intravenously until the membranes had healed . All cases were anaesthetised for repeated saline ear flushes until no further discharge was evident and no rods were seen on cytology . Topical ticarcillin and the ear cleaner were continued twice daily for 14 days after clinical resolution . The duration of treatment ranged from 14 to 36 days . Treatment was withdrawn in one case which developed a drug reaction . All other cases responded well with no adverse effects. J Clin Microbiol, 1998 May, 36(5), 1347 - 51 Persistence of a multidrug-resistant Pseudomonas aeruginosa clone in an intensive care burn unit; Hsueh PR et al.; Long-term colonization of various body sites with a multidrug-resistant Pseudomonas aeruginosa clone (resistant to piperacillin, cefoperazone, ceftazidime, aztreonam, imipenem, cefepime, cefpirome, ofloxacin, ciprofloxacin, minocycline, and aminoglycosides) with subsequent severe infections in burn patients has not been reported previously . Thirty-nine isolates of multidrug-resistant P . aeruginosa (resistant to ceftazidime and at least three of the agents listed above) recovered from various clinical samples from three patients in an intensive care burn unit from April 1997 to May 1997 and seven preserved isolates recovered from six patients in other medical wards at National Taiwan University Hospital from April 1996 to May 1997 were studied for their epidemiological relatedness . The epidemic could be attributed to a multidrug-resistant P . aeruginosa clone belonging to serogroup O:F (serogroup O:4) by means of antimicrobial susceptibility testing, O serogrouping, and analysis of the randomly amplified polymorphic DNA patterns generated by arbitrarily primed PCR of the isolates . The epidemic strain persisted in the three patients for weeks to months; in the meantime, these patients had received multiple antimicrobial agents for the management of intervening episodes of invasive infections (bacteremia, ventilator-associated pneumonia, and/or catheter-related sepsis) caused by this strain, as well as concomitant infections due to other organisms . The strain had been isolated only once previously, from a burn patient who was on the unit in December 1996 . The present report, describing a small outbreak due to P . aeruginosa, documents the fact that a single clone of multidrug-resistant P . aeruginosa can cause long-term persistence in different body sites of burn patients and that the colonization can subsequently result in various severe infections. J Immunol, 1998 May 1, 160(9), 4330 - 6 Induction of Fas ligand in murine bone marrow NK cells by bacterial polysaccharides; Halaas O et al.; Bacterial polysaccharides have a wide range of activities in mammals . We have studied the effect of LPS and poly-beta-(1-->4)-D-mannuronate (mannuronan, poly-M), an exopolysaccharide from Pseudomonas aeruginosa, on the cytotoxicity mediated by murine bone marrow cells (BMC) . Addition of LPS or mannuronan to BMC induced a time- and dose-dependent cytotoxicity against Jurkat cells . The LPS- or mannuronan-induced cytotoxicity was due to increased Fas ligand (FasL) expression by BMC, since 1) Fas-transfected L1210-Fas target cells were more susceptible to lysis than the Fas(low)-expressing parent L1210 cells, 2) stimulated BMC from FasL-defective gld/gld mice were not cytolytic and, 3) the cytolytic activity of normal BMC was inhibited by a Fas-Fc fusion protein . Flow cytometry showed an increase in surface FasL in LPS-stimulated BMC . RT-PCR analysis of BMC revealed constitutive expression of FasL mRNA, which was increased after LPS stimulation . Immunomagnetic depletion of NK1.1-, CD2-, or CD32/16-expressing cells from BMC abrogated the LPS-induced BMC cytotoxicity against L1210-Fas cells, suggesting that NK cells were the cytotoxic effector cells . Depletion of CD45R/B220-, Gr-1-, or CD11b/Mac-1-expressing cells only partially decreased BMC-mediated cytotoxicity, and depletion of CD4- or CD8a-expressing cells had no effect . The results support the conclusion that LPS and mannuronan induce expression of cytotoxic FasL on bone marrow NK cells. J Bacteriol, 1998 May, 180(9), 2306 - 11 S-layered Aneurinibacillus and Bacillus spp . are susceptible to the lytic action of Pseudomonas aeruginosa membrane vesicles; Kadurugamuwa JL et al.; When S-layered strains of Bacillus stearothermophilus and Aneurinibacillus thermoaerophilus, possessing S-layers of different lattice type and lattice constant as well as S-(glyco)protein chemistry, and isogenic S-layerless variants were subjected to membrane vesicles (MVs) from P . aeruginosa during plaque assays on plates or CFU measurements on cell suspensions, all bacterial types lysed . Electron microscopy of negative stains, thin sections, and immunogold-labelled MV preparations revealed that the vesicles adhered to all bacterial surfaces, broke open, and digested the underlying peptidoglycan-containing cell wall of all cell types . Reassembled S-layer did not appear to be affected by MVs, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the S-(glyco)proteins remained intact . meso-Diaminopimelic acid, as a peptidoglycan breakdown product, was found in all culture supernatants after MV attack These results suggest that even though MVs are much larger than the channels which penetrate these proteinaceous arrays, S-layers on gram-positive bacteria do not form a defensive barrier against the lytic action of MVs . The primary mode of attack is by the liberation from the MVs of a peptidoglycan hydrolase, which penetrates through the S-layer to digest the underlying peptidoglycan-containing cell wall . The S-layer is not affected by MV protease. Infect Immun, 1998 May, 66(5), 2170 - 9 Generation of neutralizing antipeptide antibodies to the enzymatic domain of Pseudomonas aeruginosa exotoxin A; Elzaim HS et al.; Burn patients suffer a break in the physical barrier (skin), which, when combined with their generalized state of immunodeficiency, creates an open window for opportunistic infections, mainly with Pseudomonas aeruginosa . Infection of the burn wound has always been a major factor in retardation of wound healing, and sepsis remains the leading cause of death in burn patients . Because studies have shown that topical treatment with antiexotoxin A (ETA) antibodies significantly increases survival in rats infected with toxin-producing strains of P . aeruginosa, we examined 11 synthetic peptides encompassing 12 to 45 amino acid (aa) residues, representing what were predicted by computer analysis to be the most hydrophilic and antigenic regions of ETA . These synthetic peptides were injected into rabbits for antibody production . Different groups of rabbits were immunized with a combination of peptides, with each combination representing one of the three distinct domains of ETA . Animals immunized with various peptide combinations produced peptide-specific antibodies that exhibited cross-reactivity to ETA . Two major epitopes were identified on the ETA molecule by experiments with peptide-specific antibodies in enzyme-linked immunosorbent assay and immunoprecipitation . One of these epitopes was located in the translocation domain (II) (aa 297 to 310), while the other was mapped to the last 13 aa residues at the carboxy-terminal end of the enzymatic domain (III) (aa 626 to 638) . Of these two regions, the epitope in the enzymatic domain induced a much higher level of neutralizing antibodies that abrogated the cytotoxic activity of ETA in vitro . Antibodies to this epitope blocked the ADP-ribosyltransferase activity of ETA and appeared to interfere with binding of the substrate elongation factor 2 to the enzymatic active site of