Diagn Microbiol Infect Dis, 1994 May, 19(1), 39 - 46 In vitro synergism of ceftriaxone combined with aminoglycosides against Pseudomonas aeruginosa; Laverdiere M et al.; The antipseudomonal activities of ceftriaxone (CEF) or ceftazidime (CAZ), each combined with tobramycin (TOB) or netilmicin (NET), against 90 clinically significant Pseudomonas aeruginosa isolates were examined both by checkerboard and time-kill assays . As expected, susceptibility testing of single antibiotics by agar dilution demonstrated good activity for CAZ (89% susceptible), TOB (94%), and NET (58%), but poor activity for CEF (15%) . Checkerboard studies revealed striking synergy (FIC indices < or = 0.5) for CEF, however, in combination with either TOB (72%) or NET (81%), compared with CAZ-TOB (44%) or CAZ-NET (60%) (P < 0.01, respectively) . No antagonism (FIC indices > or = 4) was found in any of these combinations . The MIC90s of CEF, CAZ, or aminoglycosides in the combinations were reduced at least fourfold: CEF, from > 128 to 32 mg/liter; CAZ, from 16 to 4 mg/liter; TOB, from 4 to 0.5 mg/liter; and NET, from 32 to 4 mg/liter . With CEF and NET, the percentage of strains sensitive to < or = 8 mg/liter of both drugs alone and in combination increased from 9% to 69% . Results of the time-kill assay for CEF-NET agreed reasonably well with the checkerboard method (Spearman rank correlation coefficient, 0.40, P < or = 0.01), and generated a bactericidal outcome in 60% (24 of the 40 isolates), when tested with combinations at 1/4 MBC of either antibiotic alone . Importantly, concentrations of CEF and aminoglycoside combinations that demonstrated synergy by either checkerboard or time-kill assays were achievable in serum clinically . These data suggest a unique interaction of CEF-aminoglycoside combinations against P . aeruginosa.(ABSTRACT TRUNCATED AT 250 WORDS) Int Arch Allergy Immunol, 1994 May, 104(1), 33 - 41 Induction of inflammatory mediator release (12-hydroxyeicosatetraenoic acid) from human platelets by Pseudomonas aeruginosa; Konig B et al.; The role of platelets in acute and chronic infection has been widely discussed in various disease processes . We studied the effects of two lipolytic enzymes (phospholipase C, lipase) secreted by Pseudomonas aeruginosa strains isolated from cystic fibrosis (CF) patients with regard to 12-hydroxyeicosatetraenoic acid (12-HETE) generation from human platelets . Both phospholipase C (PLC) and lipase were secreted into the culture supernatant at the end of the logarithmic growth phase . Indeed, only culture supernatants obtained from the late logarithmic/early stationary phase of CF strains induced the generation of 12-hydroxyeicosatetraenoic acid (12-HETE) (from 15 +/- 9 to 370 +/- 98 ng, n = 7) . Purified P . aeruginosa lipase itself generated only small amounts of 12-HETE from human platelets with a maximum of 30 +/- 7 ng/10(8) platelets at the highest concentration tested (20 nkat) . A partially purified culture supernatant from P . aeruginosa strain PAO1 containing phospholipase C and lipase, but lacking glycolipid and protease activities, induced time- and dose-dependently a significant 12-HETE generation from human platelets . Maximal 12-HETE generation was observed at the highest enzyme concentrations tested (PLC: 1.35 nkat, lipase: 3.7 nkat/10(8) platelets) . To analyze whether lipase exhibits a modulatory role on PLC-induced 12-HETE generation from human platelets we inhibited lipase activity in the P . aeruginosa partially purified culture supernatant by treatment with the lipase-specific inhibitor hexadecylsulfonylfluoride (AMSF) leaving the activity of PLC unaffected (lipase-free culture supernatant) . The capacity of lipase-free culture supernatant to induce 12-HETE generation was diminished by up to 100% depending on the PLC activity.(ABSTRACT TRUNCATED AT 250 WORDS) Zh Mikrobiol Epidemiol Immunobiol, 1994 May-Jun, (3), 86 - 8 {The lectin activity of mytilan, a bioglycan from mussels, and its effect on microbial adhesion to macroorganism cells}; Zaporozhets TS et al.; One of the mechanisms of the protective action of a bioglycan isolated from the mantle of mussels of the species Crenomytalus grayanus in bacterial infections has been studied . The preparation under study has been found to possess the activity of galactose-specific lectin and considerably decreases the adhesive properties of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa . The results of this investigation open prospects for further study of this bioglycan as an agent preventing bacterial colonization in infectious diseases. Gen Pharmacol, 1994 May, 25(3), 461 - 6 Inactivation of aminoglycosides against Pseudomonas aeruginosa by a nutrition supplementation solution; Ammash HS et al.; 1 . Possible interference of a nutritional solution (Vamin) with the activity of several aminoglycosides against Pseudomonas aeruginosa was evaluated in vitro . 2 . Inactivation in cultures of 0.75, 1.2, 2.8, 65 micrograms/ml of gentamicin, kanamycin, amikacin, streptomycin, and tobramycin was induced by the addition of 1:20 v/v of the nutritional solution . 3 . This inactivation was due to the presence of specific amino acids in the mixture . Deletions of amino acids from the media and amino acid analysis of the cellular pool revealed that valine, leucine, isoleucine, tyrosine, tryptophan, phenylalanine, cysteine, methionine, or threonine were responsible for the inactivation . 4 . The concentration of threonine decreased in kanamycin and amikacin treated cells suggesting that certain aminoglycoside antibiotics undergo a decrease in activity when sensitive Ps . aeruginosa are treated with a nutrient solution . 5 . Specific amino acids may interfere with the activity of antibiotics by circumventing their effect on amino acid biosynthesis. East Afr Med J, 1994 May, 71(5), 317 - 22 Burn injuries in Zaria: a one year prospective study; Kalayi GD; Fifty three patients admitted for burn care during a 12 month period from September 1987 to August 1988 were prospectively studied . There were 36 males (60%) and 21 females (40%) with ages ranging from 3 months to 60 years . Children aged 0-4 constituted 40% but 32 (60%) were younger than 16 years . Flame burns affected 26 (49%) patients, scalds in 22 (12%), electrical burns affected four patients and chemical burn was in one . Scald was the commonest injury among children aged 0-4 (70%) . Flame, affected 33% of those aged 16 and above . Clothing fire was the commonest flame injury and it was a cause of very extensive injury (mean % BSA 45) . Kerosene burn, gas and clothing burns caused the most extensive injury with a mean % BSA 46, 41 and 45 respectively . The commonest complication was burn wound sepsis most frequently by a gram-negative bacilli (65.63%) of which Pseudomonas aeruginosa were that commonest organisms . Pseudomonas and Staphylococcus aureus were about same frequency . Duration of hospital stay ranged from 6 days to 300 days with a mean of 46.52 days . 8 patients absconded, two were transferred to a hospital near their home and 9 died, giving a mortality rate of 17% . Since burn injuries are largely preventable, it is important to define clearly the social, cultural and economic factors which contribute to burn causation in order to combat them effectively. J Formos Med Assoc, 1994 May, 93(5), 421 - 8 Hematophagic histiocytosis: a clinicopathologic analysis of 23 cases with special reference to the association with peripheral T-cell lymphoma; Chang CS et al.; The clinicopathologic features of 23 patients with hematophagic histiocytosis (HH) are described . All of them exhibited increased histiocytes associated with hemophagocytosis in the marrow . The patients usually presented with fever, hepatosplenomegaly, lymphadenopathy, and cytopenia . The underlying illnesses were heterogeneous, including non-Hodgkin's lymphoma in 17, systemic lupus erythematosus in one, diabetes mellitus in one, acute myelomonocytic leukemia in one, myelodysplastic syndrome in one, and unknown cause in two . Among 17 non-Hodgkin's lymphoma, 14 were peripheral T-cell lymphoma, two were B-cell lymphoma, and one was an undefined phenotype . Among 14 patients with peripheral T-cell lymphoma, six of the patients had nasal T-cell lymphoma . Five of these 14 patients initially diagnosed as malignant histiocytosis turned out to be T-lineage lymphoma after immunophenotypic studies . Active infections, most of viral origin, were documented in eight patients, including Epstein-Barr virus in three, cytomegalovirus in three, herpes simplex virus in three, Pseudomonas aeruginosa in one, Bacteroides vulgatus in one, and mycoplasma in one . Some of them had mixed virus and bacteria infection . Sixteen (70%) of our patients died of their acute illness within 10 weeks of the diagnosis of HH . In the past, the clinical and histologic differentiation between hematophagic histiocytosis and true histiocytic neoplasm (histiocytic medullary reticulosis/malignant histiocytosis) has proved difficult, but now these can be distinguished with immunohistologic, immunogenetic, and cytogenetic studies, especially in the cases of peripheral T-cell lymphoma with hemophagocytic syndrome. J Hosp Infect, 1994 May, 27(1), 49 - 60 Lack of association between clinical and environmental isolates of Pseudomonas aeruginosa in hospital wards; Orsi GB et al.; Seventy-three environmental and clinical isolates of Pseudomonas aeruginosa recovered from a single hospital over a 6-month period were compared for epidemiological type characteristics . Environmental isolates were obtained from sinks, taps and water, in rooms where patients were treated . The strains represented only six O-antigenic types and 8.2% of them were not typable . Serotype 011 was most frequent in the environment, whereas serotypes 06, 012 and 02,5 predominated among clinical isolates . More than 60% of all isolates belonged to four pyocin types (1, 10, 33 and 45), and approximately 80% were phage typable . Environmental isolates were more sensitive to antibiotics than clinical isolates . There was little correspondence between the types of strains of P . aeruginosa isolated from patients and those isolated from the environment . However, isolates of identical type were frequently recovered from different patients within the same clinic and were found to be related in time and location . We conclude that the environment was not an important source of P . aeruginosa infection and that transfer of organisms was mainly from patient-to-patient. J Med Microbiol, 1994 May, 40(5), 313 - 22 Genetic heterogeneity of Pseudomonas aeruginosa clinical isolates revealed by esterase electrophoretic polymorphism and restriction fragment length polymorphism of the ribosomal RNA gene region; Picard B et al.; The intra-species differentiation of Pseudomonas aeruginosa was analysed by comparing the polymorphism of esterases by conventional polyacrylamide-agarose gel electrophoresis, the physicochemical properties of the variants of the major esterase P3 and the restriction fragment length polymorphism of ribosomal RNA gene regions (ribotyping) to O-serotyping for several panels of strains selected from among a series of 257 clinical isolates and two references strains, (ATCC nos . 10145 and 27853) . The electrophoretic variation of four main kinds of esterase (P1-P4) and 11 additional esterases distinguished by their spectra of hydrolytic activity with synthetic substrates and by their sensitivity to di-isopropylfluorophosphate, allowed the discrimination of 67 zymotypes . Thirty-two esterase P3 variants were characterized by their pI, electrophoretic mobilities and titration curve analyses . They were distributed into two groups which, by these molecular criteria, seem to be distantly related . Combination of the patterns resulting from HindIII, EcoRI and BclI restriction endonuclease digestions allowed the discrimination of 33 ribotypes among 134 strains . The strains exhibiting esterase P3 variants of group 2 presented a distinct ribotype and belonged to serotype (O)12 . They could constitute a distinct group within the species . For the majority of the strains, the absence of correlation between zymotype, ribotype and serotype argues for a high level of heterogeneity within P . aeruginosa and indicates that the parallel use of the first two methods represent a potential tool for epidemiological study. Zhonghua Zheng Xing Shao Shang Wai Ke Za Zhi, 1994 May, 10(3), 218 - 21 {The relationship between opportune administration of antibiotics and infection after burn injury: an experimental study}; Xu M et al.; 45 male rabbits with 15% III degree burn were infected with Pseudomonas aeruginosa . They were divided into three groups: the prevention group, in which cpz (cefoperazone) was immediately administered after the scald at the dose of 25 mg/kg with an interval of 1/12 h for a total of 4 doses; the treatment group, in which cpz was administered 48 th after the scald with the dosage same as above; control group in which cpz was not administered . The findings were as follows: In the prevention group, gross observation showed dessication and crustation of the surface of the wound within 1 week after the scald . By employing immunofluorescent antibody it was found that P . A existed only in the superficial layer . However, ulceration and exudation were found in the control and treatment group within 3 or 4 days after the scald, and P . A was shown to have invaded the deep muscular tissue to cause intermuscular septal abscess . The positive rate of blood culture of bacteria in the prevention group (10/81) was significantly lower than that of the treatment (15/57) and the control groups (21/76, P < 0.05) . The death rate of the prevention group (2/15) was significantly lower than that of the treatment (10/15) and the control groups (9/15, P < 0.05) . The above finding showed that antibiotics specific to the infectious organism of the wound in extensive burn patients should be given in the early exudation stage in order to ward off invasive infection. Pathol Biol (Paris), 1994 May, 42(5), 505 - 9 {Production of elastase, exotoxin A and alkaline protease during bronchopulmonary exacerbations in patients with mucoviscidosis chronically infected by Pseudomonas aeruginosa}; Jaffar-Bandjee MC et al.; The authors have studied the production of exoproteins by Pseudomonas aeruginosa in the sputa of 18 patients suffering from cystic fibrosis, during 29 bronchopulmonary exacerbations and also after the recovery of a stable state . Significant levels of exoproteins were detected but with a large heterogenity of intra and inter individual variations . A significant decrease in the production of the three exoproteins was found after twelve days of antibiotherapy, without any correlation between exoprotein levels and colony forming units in the sputa . During the intercrisis phase, exoproteins levels were practically undetectable . These facts and the good correlation between clinical symptoms support the hypothesis of a renewal of virulence of Pseudomonas aeruginosa during these periods of bronchopulmonary exacerbation in cystic fibrosis. Pathol Biol (Paris), 1994 May, 42(5), 491 - 7 {Epidemiological studies of the susceptibility of Pseudomonas aeruginosa to antibiotics}; Bert F et al.; The susceptibility to antibiotics of 1367 non-replicate strains of Pseudomonas aeruginosa isolated at Beaujon Hospital between 1990 and 1992 was investigated and compared with the serogroup O and the strain origin (ward, sample) . Five betalactam resistance patterns were distinguished according to susceptibility to ticarcillin, piperacillin, ceftazidime and aztreonam,: 1 = SSSS, 2 = RRSS, 3 = RRRR, 4 = RSSR, 5 = RRSR . The other antibiotics studied were imipenem, tobramycin, amikacin, ciprofloxacin and fosfomycin . Resistance to all antibiotics, fosfomycin excepted, was higher in intensive care units than in other wards . The respective frequencies of the phenotypes were: 70.3%, 4.3%, 11.8%, 10.2% and 3.4% . The frequency of pattern 3 steadily increased between 1990 and 1992 at the expense of pattern 1, whereas patterns 2, 4 and 5 remained stable . The most common serogroups were O6 (15.8%), O11 (14.5%) and O1 (9.9%) . The O11 strains were more widespread in intensive care units than in other wards and were more resistant to antibiotics . Most of the O12 strains displayed pattern 2 and were highly resistant to antibiotics. Ukr Biokhim Zh, 1994 May-Jun, 66(3), 40 - 4 {Fatty acids of variants of the strain Pseudomonas aeruginosa 1C-- destroyer of alkylsulfates}; Kryvets' IO et al.; The differences in the fatty acid composition of morphological variants of strain-destroyer of sodium dodecyl sulfate (SDS) Pseudomonas aeruginosa 1C are shown . Morphological variants differ by the degree of resistance against SDS and by the level of the destructive activity . The abnormally high level of fatty acids unsaturation is the particularity of their composition of the resistant smooth variant . The intermediate filamentous variant synthesizes the acid with 19 atoms of carbon absent in other variants . The rough variant has the least various spectrum of fatty acids . In the stationary phase the cells of the rough variant contain the largest number of cyclopropane acids. Shock, 1994 May, 1(5), 343 - 6 Release of endothelin in relation to tumor necrosis factor-alpha in porcine Pseudomonas aeruginosa-induced septic shock; Han JJ et al.; Septic shock is characterized by surges of tumor necrosis factor-alpha (TNF-alpha) along with myocardial dysfunction and systemic hypotension . TNF-alpha promotes the release of immunoreactive endothelin (ET) . Because TNF-alpha is elevated in septic shock, we hypothesized that elevated levels of endothelin can contribute to cardiac dysfunction and hypotension . We infused live Pseudomonas aeruginosa into anesthetized, hemodynamically monitored young swine and measured ET and TNF-alpha . Septic swine developed systemic arterial hypotension and had significantly elevated TNF-alpha (4.15 +/- .41 U/ml at 1 h versus .40 +/- .13 U/ml at time zero) compared to control animals . ET levels were significantly elevated at 4 h (52.38 +/- 12.88 pg/ml vs . 10.45 +/- 1.82 pg/ml at time zero) and correlated negatively with the decline in cardiac output . We then passively immunized swine using anti TNF-alpha prior to the induction of sepsis to examine if TNF played a central role in the release ET . The anti TNF-alpha effectively removed circulating TNF-alpha bioactivity in septic animals . Anti-TNF-alpha-treated animals did not develop significant systemic arterial hypotension and had significant attenuation in endothelin (19.01 +/- 4.18 pg/ml at 4 h compared to 52.38 +/- 12.88 pg/ml in septic animals at 4 h) which correlated with preservation of cardiac output . TNF-alpha may cause cardiac dysfunction in sepsis syndrome through increased release of ET. Electrophoresis, 1994 May, 15(5), 594 - 6 Rapid method for isolating detergent-insoluble outer membrane proteins from Pseudomonas aeruginosa; Ravaoarinoro M et al.; The present study compares two techniques for isolating outer membrane proteins (OMPs) of Pseudomonas aeruginosa, Method A - selective solubilization with sodium lauryl sarcosinate, and Method B - isopycnic sucrose gradient centrifugation, using three criteria: the amount of proteins obtained, polypeptide patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their practical aspects . Method A appears to be superior to Method B . It yields a higher outer membrane protein content and a similar polypeptide pattern as Method B, but is more rapid and less cumbersome. Hum Gene Ther, 1994 May, 5(5), 615 - 39 Gene therapy for cystic fibrosis using E1-deleted adenovirus: a phase I trial in the nasal cavity . The University of North Carolina at Chapel Hill; Boucher RC et al.; Cystic fibrosis (CF) is an autosomal recessive disease that reflects mutations in the CFTR gene . Multiple mutations in this gene have been detected that lead to a protein (CFTR) that is abnormally metabolized, dysfunction, or both . The full spectrum of the activities of the gene product have not been defined, but it is clear that CFTR can act as a cAMP-regulated Cl- channel . This type of defect is consistent with the physiologic characterization of CF epithelia, which has revealed abnormalities in salt and water transport . In the lung, abnormalities in epithelial salt and water metabolism lead to abnormal mucociliary clearance . This defect in clerance represents a major failure of lung defense and leads ultimately to infection of the lung with Staphylococcus aureus, Pseudomonas aeruginosa, and other bacterial organisms . The chronic inflammatory response to this persistent intraluminal bacterial infection leads to protease-induced destruction of airway walls and finally, lung failure . More than 95% of CF patients die of lung disease . The clinical therapy of CF lung disease is limited to agents designed to promote clearance of secretions from the lung and antibiotics to treat the chronic bacterial infection . Recent laboratory demonstrations that introduction of the normal CFTR cDNA into CF cells corrects the ion transport defects of these cells has led to the hypothesis that gene therapy in the lung can be an effective, novel mode of therapy for this lung disease . The classic gene transfer vectors, e.g., retroviruses, appear to be not well suited for therapy of lung disease because of the low proliferation rate of airway epithelia in vivo . Recently, adenoviruses, which have a natural tropism for airway epithelia, have been genetically modified (E1-deleted) in an attempt to reduce potential toxicity of this virus and provide space for the CFTR cDNA . A series of in vitro studies have shown that this vector is highly efficient for transferring CFTR into airway epithelial cells in culture and correcting the CF defect . Further, studies in whole animals appear to indicate that this mode of gene transfer is associated with a low degree of toxicity . The present study is a dose-effect study designed to test for the safety and efficacy of E1-deleted recombinant adenovirus containing the CFTR cDNA under a CMV-beta-actin promoter in CF nasal epithelia.(ABSTRACT TRUNCATED AT 400 WORDS) J Trauma, 1994 May, 36(5), 714 - 8; discussion 718-9 Effect of inhibiting leukocyte integrin (CD18) and selectin (L-selectin) on susceptibility to infection with Pseudomonas aeruginosa; Garcia NM et al.; Leukocyte (WBC) adherence to endothelial cells has been implicated in the pathogenesis of microvascular injury . The process of leukocyte adherence is mediated by both the integrin and selectin families of molecules, and their interaction with specific endothelial ligands . Antibodies directed against the leukocyte integrin CD18 and L-selectin have been developed and functionally inhibit leukocyte adherence in models of inflammatory injury . We asked the question: Does inhibition of leukocyte adherence by administration of monoclonal antibody directed against either CD18, integrins (R15.7, R7.1) or against L-selectin (DREG 200) increase susceptibility to infection? New Zealand white rabbits were shaved and injected subcutaneously on their dorsum with Pseudomonas aeruginosa (ATCC#27853) at two sites each of 10(8) and 10(7) colony forming units . Animals were monitored with daily determination of weight, temperature, WBC counts, hematocrit, and killed at 1 week for determination of abscess formation . There were four blinded experimental groups: (1) Saline (2 mL/kg); (2) DREG 200 (2 mg/kg); (3) R7.1 (2 mg/kg); or (4) R15.7 (2 mg/kg) . At the 10(7) and 10(8) injection sites the R15.7 group had an increased rate and size of abscess formation compared with controls . The R7.1 group had an increased rate at the 10(8) injection site . There was no significant difference in the percentage of the abscess formation or mean area between the controls and DREG 200-treated groups . We conclude that giving antibody to CD18 increased susceptibility to infection while giving antibody to L-selectin does not. Science, 1994 Apr 15, 264(5157), 382 - 8 Prevention of drug access to bacterial targets: permeability barriers and active efflux; Nikaido H; Some species of bacteria have low-permeability membrane barriers and are thereby "intrinsically" resistant to many antibiotics; they are selected out in the multitude of antibiotics present in the hospital environment and thus cause many hospital-acquired infections . Some strains of originally antibiotic-susceptible species may also acquire resistance through decreases in the permeability of membrane barriers . Another mechanism for preventing access of drugs to targets is the membrane-associated energy-driven efflux, which plays a major role in drug resistance, especially in combination with the permeation barrier . Recent results indicate the existence of bacterial efflux systems of extremely broad substrate specificity, in many ways reminiscent of the multidrug resistance pump of mammalian cells . One such system seems to play a major role in the intrinsic resistance of Pseudomonas aeruginosa, a common opportunistic pathogen . As the pharmaceutical industry succeeds in producing agents that can overcome specific mechanisms of bacterial resistance, less specific resistance mechanisms such as permeability barriers and multidrug active efflux may become increasingly significant in the clinical setting. J Biol Chem, 1994 Apr 8, 269(14), 10431 - 7 Cloning the structural gene for the 49-kDa form of exoenzyme S (exoS) from Pseudomonas aeruginosa strain 388; Kulich SM et al.; We report the purification and proteolytic characterization of the 49-kDa form of exoenzyme S and the cloning of the structural gene for the 49-kDa form of exoenzyme S (exoS) . The 49-kDa form of exoenzyme S was purified from SDS-polyacrylamide gels . Conditions were established that allowed efficient trypsin digestion of the 49-kDa form of exoenzyme S . Amino acid sequence determination of the amino terminus and tryptic peptides of the 49-kDa form of exoenzyme S allowed the generation of degenerate oligonucleotides, which were used to amplify DNA encoding an amino-terminal sequence and an internal sequence of the 49-kDa form of exoenzyme S . These DNA fragments were used to clone the entire structural gene for the 49-kDa form of exoenzyme S (exoS) from a cosmid library of Pseudomonas aeruginosa strain 388 . The 49-kDa form of exoenzyme S (ExoS) is predicted to be a 453 amino acid protein . The predicted amino acid sequence indicates that ExoS is secreted from Pseudomonas without cleavage of an amino-terminal sequence . BESTFIT analysis identified three regions of alignment between ExoS and the active site of Escherichia coli heat-labile enterotoxin . One region of homology appears to be shared among several members of the family of bacterial ADP-ribosyltransferases. Genitourin Med, 1994 Apr, 70(2), 127 - 9 Infectious osteitis pubis in an HIV seropositive female; Desmond N et al.; We report a case of infectious osteitis pubis following a first trimester abortion in a female seropositive for the human immunodeficiency virus (HIV) . Joint aspiration yielded Pseudomonas aeruginosa and the patient was successfully treated with oral ciprofloxacin. J Antibiot (Tokyo), 1994 Apr, 47(4), 477 - 86 Studies on beta-lactam antibiotics . IV . An improved synthesis of 3-(isothiazolylthiomethyl)cephalosporins and its application to new derivatives; Hara R et al.; An improved synthesis and in vitro activity of cephalosporins with a (4-carboxy-3-hydroxy-5-isothiazolyl)thiomethyl group at the 3-position and its application to the preparation of new derivatives are described . These compounds showed excellent activity against Gram-negative bacteria including beta-lactamase producing strains . Among them, 2f was the most interesting because of its broad spectrum of antibacterial activity, including Gram-positive bacteria, and its outstanding inhibitory potency against Pseudomonas aeruginosa. Br J Plast Surg, 1994 Apr, 47(3), 158 - 61 An evaluation of a new lactic acid polymer drug delivery system: a preliminary report; Sawada Y et al.; A new drug delivery system, made of a mixture of DL-lactic acid polymer and a copoly (L-lactic acid/delta-valerolactone) polymer (LAP) containing varying concentrations of Ofloxacin, has been evaluated . Results revealed that a LAP drug delivery system containing 0.03% Ofloxacin inhibited the in vitro growth of clinically derived Staphylococcus aureus and Pseudomonas aeruginosa . Further, in an in vivo situation in rats, at 1 week after the wound was inflicted, no infection was macroscopically seen in split-thickness wounds that had been covered by LAP drug delivery system containing 0.5 or 0.03% Ofloxacin, and more than 80% of the wound area had epithelialised. Jikken Dobutsu, 1994 Apr, 43(2), 191 - 7 {Elimination of Pseudomonas aeruginosa from an experimental nude mouse colony}; Nakamura N et al.; In order to eliminate Pseudomonas (P.) aeruginosa from a contaminated nude mouse colony, the following procedures were carried out: improvement of environmental sanitation using an effective disinfectant against the organism; supply of tap water acidified with hydrochloric acid at pH2.5-3.0; elimination of mice positive in isolation of P . aeruginosa from the nude mouse colony . The experimental results indicated that P . aeruginosa were successfully eliminated from the colony though a combination of the above three procedures. Eur J Biochem, 1994 Apr 1, 221(1), 555 - 61 Catabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa . Changes of allosteric properties resulting from modifications at the C-terminus; Tricot C et al.; Ornithine carbamoyltransferases (OTCases) catalyse the formation of citrulline and phosphate from ornithine and carbamoylphosphate by a thermodynamically favoured reaction . In vivo, catabolic OTCase of Pseudomonas aeruginosa promotes the reverse reaction, the phosphorolysis of citrulline . Although the enzyme is assayed in vitro in the direction of citrulline synthesis, the enzyme cannot perform this reaction in vivo due to poor affinity for carbamoylphosphate and high cooperativity towards this substrate . Furthermore, the dodecameric catabolic OTCase is an allosteric enzyme; the enzyme is stimulated by nucleoside monophosphates and inhibited by polyamines (e.g . spermidine) . A previous study showed that a modification of the C-terminus of the catabolic OTCase alters the homotropic cooperativity of the enzyme . We have now investigated the importance of the C-terminus for homotropic and heterotropic cooperativity by site-directed mutagenesis . Deletion of the C-terminal Ile335 residue strongly reduced cooperativity for carbamoylphosphate and sensitivity to spermidine . These properties were essentially restored when the two C-terminal amino acids (Asp334 and Ile335) were removed by deletion . However, in this variant enzyme, AMP failed to abolish carbamoylphosphate cooperativity completely, whereas the wild-type enzyme was rendered virtually non-cooperative by AMP . An extension of catabolic ornithine carbamoyltransferase by 15 amino acid residues interfered with both homotropic and heterotropic interactions and lowered the maximal velocity . All variant enzymes had the same dodecameric structure as the wild type and differed only slightly in affinity for the second substrate ornithine . A structural model of the dodecamer, at 0.3-nm resolution, suggests that the C-terminus could be involved in trimer/trimer interaction . We propose that modifications at the C-terminus alter the trimer/trimer interface and, in addition, removes the salt bridge His5-Ile335 within a monomer . These changes profoundly and indirectly modify the allosteric transition and consequently the interactions of the dodecamer with carbamoylphosphate and effectors. Surgery, 1994 Apr, 115(4), 480 - 7 Liposome encapsulation: a method for enhancing the effectiveness of local antibiotics; Price CI et al.; BACKGROUND . Treatment of contaminated surgical wounds is often complicated by the failure of local or systemic antibiotic treatment and prophylaxis . Locally administered liposome-encapsulated antimicrobials may offer advantages over free antibiotics, including an increase in efficacy, ease of administration, and safety . The therapeutic advantages, as well as the absorption and distribution of locally administered liposome-encapsulated antibiotics, were compared with those of locally applied unencapsulated antibiotics in a contaminated wound model . METHODS . Anesthetized rats had a 1 cm incision over the midback that was inoculated with 10(8) colony-forming units Pseudomonas aeruginosa (group 1; n = 102) or left uninfected (group 2; n = 35) . Before wound closure, infected animals were treated with a local application of 0.3 ml saline solution (untreated; n = 30), 5.5 mg tobramycin in 0.3 ml saline solution (free tobramycin; n = 30), or 0.3 ml liposome-encapsulated tobramycin (LET; n = 42) . Animals were killed 24, 48, and 72 hours after operation; serum and tissue tobramycin concentrations and tissue quantitative cultures were studied . Liposomes were radiolabeled to examine organ distribution . RESULTS . The data show that LET produced sustained local concentrations of antibiotic compared with free drug; sustained concentration prolonged the antimicrobial effect despite a single dose of antibiotic . LET reduced tissue bacterial counts to a greater extent and for a longer period of time than free tobramycin . The presence of infection further reduced clearance of LET from the infected site . CONCLUSIONS . The liposomal delivery of local antibiotics in this model of surgical wound infection reduced the number of organisms more effectively than locally applied free drug . Animals treated with LET had consistently less than the 10(5) organisms per gm tissue considered critical for invasive infection, suggesting that liposomal antibiotics may be clinically useful in surgical wound prophylaxis. J Bacteriol, 1994 Apr, 176(8), 2184 - 93 Cloning and nucleotide sequence of the glpD gene encoding sn-glycerol-3-phosphate dehydrogenase of Pseudomonas aeruginosa; Schweizer HP et al.; Nitrosoguanidine-induced Pseudomonas aeruginosa mutants which were unable to utilize glycerol as a carbon source were isolated . By utilizing PAO104, a mutant defective in glycerol transport and sn-glycerol-3-phosphate dehydrogenase (glpD), the glpD gene was cloned by a phage mini-D3112-based in vivo cloning method . The cloned gene was able to complement an Escherichia coli glpD mutant . Restriction analysis and recloning of DNA fragments located the glpD gene to a 1.6-kb EcoRI-SphI DNA fragment . In E . coli, a single 56,000-Da protein was expressed from the cloned DNA fragments . An in-frame glpD'-'lacZ translational fusion was isolated and used to determine the reading frame of glpD by sequencing across the fusion junction . The nucleotide sequence of a 1,792-bp fragment containing the glpD region was determined . The glpD gene encodes a protein containing 510 amino acids and with a predicted molecular weight of 56,150 . Compared with the aerobic sn-glycerol-3-phosphate dehydrogenase from E . coli, P . aeruginosa GlpD is 56% identical and 69% similar . A similar comparison with GlpD from Bacillus subtilis reveals 21% identity and 40% similarity . A flavin-binding domain near the amino terminus which shared the consensus sequence reported for other bacterial flavoproteins was identified. J Med Microbiol, 1994 Apr, 40(4), 282 - 7 Characterisation of the external surfaces of Pseudomonas aeruginosa isolated from human blood and respiratory tract; Misumi H et al.; The surface structures of the cell envelopes of 16 clinical isolates of Pseudomonas aeruginosa were examined by electronmicroscopy with the new fixation technique of freeze-substitution . Two types of structures were observed among the organisms . In one group of strains, mostly isolated from blood, a dense fibrous layer c . 30 nm thick was found around the outer-membrane surface, whereas no such structure was observed in the other group of isolates, most of which were from sputum . Lipopolysaccharides extracted from the isolates with a dense fibrous layer were found by SDS-PAGE to have long O-polysaccharide chains, whereas strains without such a layer mostly had lipopolysaccharides that lacked high mol . wt . O-polysaccharide chains. J Med Microbiol, 1994 Apr, 40(4), 275 - 81 Differentiation of Pseudomonas aeruginosa strains by ribotyping: high discriminatory power by using a single restriction endonuclease; Grattard F et al.; The genotypic diversity of 40 presumably epidemiologically unrelated strains of Pseudomonas aeruginosa belonging to nine different O-serotypes was analysed according to ribosomal DNA fingerprints . Ribotyping was performed with a digoxigenin-labelled DNA probe and four restriction endonucleases . Characteristic banding patterns of three to 12 bands were obtained with the different endonucleases . Among the 40 strains, eight, nine, 10 and 29 different ribotypes were differentiated with EcoRI, the combination EcoRI+HindIII, BamHI and PvuII, respectively . Poor correlations were noted between the results of serotyping and those of ribotyping . With the latter method, indices of discrimination were calculated for each enzyme from the data of the 40 unrelated strains: the values ranged from 0.678 for EcoRI to 0.979 for PvuII . Epidemiologically related samples were also tested; this enabled assessment of whether the method was able to cluster strains from a common origin with each of the enzymes tested . Ribotyping with PvuII endonuclease is proposed for screening large numbers of P . aeruginosa strains in epidemiological studies . Additional enzymes could be used to further increase the discrimination between isolates found to be indistinguishable with PvuII enzyme. J Bacteriol, 1994 Apr, 176(7), 1821 - 30 Pseudomonas aeruginosa AlgG is a polymer level alginate C5-mannuronan epimerase; Franklin MJ et al.; Alginate is a viscous extracellular polymer produced by mucoid strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis . Alginate is polymerized from GDP-mannuronate to a linear polymer of beta-1-4-linked residues of D-mannuronate and its C5-epimer, L-guluronate . We previously identified a gene called algG in the alginate biosynthetic operon that is required for incorporation of L-guluronate residues into alginate . In this study, we tested the hypothesis that the product of algG is a C5-epimerase that directly converts D-mannuronate to L-guluronate . The DNA sequence of algG was determined, and an open reading frame encoding a protein (AlgG) of approximately 60 kDa was identified . The inferred amino terminus of AlgG protein contained a putative signal sequence of 35 amino acids . Expression of algG in Escherichia coli demonstrated both 60-kDa pre-AlgG and 55-kDa mature AlgG proteins, the latter of which was localized to the periplasm . An N-terminal analysis of AlgG showed that the signal sequence was removed in the mature form . Pulse-chase experiments in both E . coli and P . aeruginosa provided evidence for conversion of the 60- to the 55-kDa size in vivo . Expression of algG from a plasmid inan algG (i.e., polymannuronate-producing) mutant of P . aeruginosa restored production of an alginate containing L-guluronate residues . The observation that AlgG is apparently processed and exported from the cytoplasm suggested that it may act as a polymer-level mannuronan C5-epimerase . An in vitro assay for mannuronan C5 epimerization was developed wherein extracts of E . coli expressing high levels of AlgG were incubated with polymannuronate . Epimerization of D-mannuronate to L-guluronate residues in the polymer was detected enzymatically, using a L-guluronate-specific alginate lyase of Klebsiella aerogenes . Epimerization was also detected in the in vitro reaction between recombinant AlgG and poly-D-mannuronate, using high-performance anion-exchange chromatography . The epimerization reaction was detected only when acetyl groups were removed from the poly-D-mannuronate substrate, suggesting that AlgG epimerization activity in vivo may be sensitive to acetylation of the D-mannuronan residues . These results demonstrate that AlgG has polymer-level mannuronan C5-epimerase activity. Infect Immun, 1994 Apr, 62(4), 1320 - 7 lasA and lasB genes of Pseudomonas aeruginosa: analysis of transcription and gene product activity; Toder DS et al.; The lasA gene was the first of the Pseudomonas aeruginosa genes involved in proteolysis and elastolysis to be cloned and sequenced . Its function and significance have been studied by genetic approaches (D . S . Toder, M . J . Gambello, and B . H . Iglewski, Mol . Microbiol . 5:2003-2010, 1991) and by attempts to purify an active fragment of the protein (J . E . Peters and D . R . Galloway, J . Bacteriol . 172:2236-2240, 1990) . To further study LasA in vivo, we have constructed and characterized an insertional mutant in the lasA gene in strain PAO1 (PAO-A1) and in the lasB insertional mutant, PAO-B1 . Analysis of these isogenic strains demonstrates that the lasA lesion diminished elastolysis more than proteolysis and that LasA is required for staphylolytic activity . Despite previous suggestions that lasB elastase cleaves the LasA protein, the size of the LasA protein was the same whether or not lasB elastase was present . Expression of lasA in a lasR-negative mutant, PAO-R1, demonstrated that the LasA protein is produced in an active form in the absence of (lasB) elastase or alkaline protease and is itself a protease with elastolytic activity . We also observed that PAO-A1 was closer to the parental phenotype, with respect to elastolytic and proteolytic activities, than the previously characterized, chemically induced lasA mutant PAO-E64 . Quantification of promoter activity with lasA::lacZ and lasB::lacZ fusions suggests that PAO-E64 harbors a mutation in a gene which regulates expression of both lasA and lasB. Infect Immun, 1994 Apr, 62(4), 1137 - 43 Specificity and function of murine monoclonal antibodies and immunization-induced human polyclonal antibodies to lipopolysaccharide subtypes of Pseudomonas aeruginosa serogroup 06; Pier GB et al.; Structural and antigenic heterogeneity has been noted among lipopolysaccharides (LPS) produced by Pseudomonas aeruginosa within serogroups previously considered to be serologically homogeneous . We characterized murine monoclonal antibodies (MAbs) and immunization-induced human polyclonal antibodies reactive with one or more of five structurally variant LPS subtypes belonging to serogroup 06 of the International Antigenic Typing System . Analyses of five different MAbs employing purified LPS or whole patterns of subtype specificity, ranging from recognition of a single subtype to reactivity with all five . MAb-mediated opsonophagocytic killing and in vivo protection against live challenge in mice correlated, in general, with differential binding to various LPS subtypes . In comparison, sera from human vaccinees immunized with LPS-derived high-molecular-weight polysaccharide from P . aeruginosa Fisher immunotype 1, one of five serogroup 06 subtypes, exhibited LPS binding and opsonic activity against all five subtypes . Antibodies in the human sera effectively inhibited binding to all five LPS subtype antigens of the cross-reactive MAb, LC3-2H2, suggesting the existence of a common serogroup-related epitope . These findings emphasize the importance of defining subtype-associated variations in LPS antigenicity and corresponding differences in antibody specificity and function as a basis for designing immunoprophylactic or therapeutic strategies which target P . aeruginosa LPS. J Chemother, 1994 Apr, 6(2), 111 - 6 Occurrence and antibiotic-resistance of Pseudomonas species isolated from drinking water in southern Greece; Papapetropoulou M et al.; A total of 194 samples of drinking waters consisting of 88 tap waters and 106 non-carbonated bottled waters were processed for isolation of Pseudomonas species during a 4-month period according to standard methods . Pseudomonas aeruginosa was the predominant isolated Pseudomonas species . Twenty-eight (14.4%) P . aeruginosa were isolated from 194 samples . Eight (9%) were isolated from 88 tap water samples and 20 (18.8%) from 106 bottled water samples . Eight (9%) tap waters yielded non-P . aeruginosa strains while bottled waters yielded 22 (20.7%) non-P . aeruginosa strains (P < 0.05) . Antibiotic-resistant strains of Pseudomonas species have been isolated from the drinking waters . All but Pseudomonas stutzeri species had a multiple chloramphenicol-erythromycin resistance phenotype . Streptomycin and tetracycline resistance for P . aeruginosa was invariably accompanied by chloramphenicol, tetracycline, erythromycin and nalidixic acid resistance . The susceptibility of Pseudomonas species to newer antimicrobial agents (beta lactams, aminoglycosides, third generation cephalosporins and quinolones) was also evaluated . Ceftazidime and ciprofloxacin seemed to be the most active molecules . There were no resistant P . aeruginosa and P . stutzeri strains to all newer antibiotics tested while Pseudomonas maltophilia was the most resistant among the tested species (69.2% resistance for the newer antibiotics). Eur J Clin Microbiol Infect Dis, 1994 Apr, 13(4), 315 - 8 Imipenem and meropenem induced resistance to beta-lactam antibiotics in Pseudomonas aeruginosa; Giacometti A et al.; The ability of imipenem and meropenem in subinhibitory concentrations to influence the results of disk diffusion susceptibility tests was assessed . Selection of stably derepressed mutants resistant to beta-lactam antibiotics other than carbapenems was also investigated . Beta-lactams were shown to be subject to carbapenem-mediated antagonism in the disk diffusion test . On the other hand in vitro selection of stably derepressed mutants resistant to other beta-lactams could not be demonstrated. J Antimicrob Chemother, 1994 Apr, 33(4), 765 - 75 The effect of erythromycin on Pseudomonas aeruginosa and neutrophil mediated epithelial damage; Tanaka E et al.; Erythromycin therapy for long periods may benefit patients with chronic bronchial sepsis colonized by Pseudomonas aeruginosa despite the lack of antibacterial activity . We have investigated the effect of filtrates of 24 h P . aeruginosa cultures (CF) with or without erythromycin 0.5, 5, 20 mg/L on human nasal epithelium in the absence or presence of polymorphonuclear leucocytes (PMN) . Ciliary beat frequency (CBF) and epithelium integrity were examined for 4 h . Erythromycin (20 mg/L) alone did not affect epithelium . CF without erythromycin slowed CBF by 63.5% of control at 4 h, and caused disruption of surface integrity in 80% of the epithelium . Addition of erythromycin to CF did not inhibit these effects . Erythromycin did not affect growth of P . aeruginosa . Filtrates of P . aeruginosa cultured with erythromycin (5 and 20 mg/L) caused less CBF slowing (37.2% and 19.2% of control, respectively) and epithelial disruption (4.2% and 6.7%, respectively) . Unstimulated PMN (10(7)/mL) slowed CBF by 13% of control at 4 h but did not cause epithelial disruption . PMN and CF together slowed CBF (95.4% of control) and damaged epithelium (93.3% of epithelium disrupted) synergically . Pre-incubation of PMN with erythromycin did not inhibit these effects . PMN and filtrates of P . aeruginosa cultured with erythromycin (5 and 20 mg/L) caused less CBF slowing (58.0% and 33.6% of control, respectively) and epithelial disruption (40.0% and 13.3%, respectively) . Erythromycin may benefit patients by reducing P . aeruginosa production of factors which damage epithelium and stimulate neutrophil mediated cytotoxicity. Can J Microbiol, 1994 Apr, 40(4), 292 - 7 Urokinase enhances the growth of Pseudomonas spp . in vitro under nonshaking (oxygen limited) conditions; Hart DA et al.; Urokinase is a proteinase that normally functions as a plasminogen activator . It is detected in a number of tissues and can be expressed by inflammatory cells such as macrophages and polymorphonuclear leucocytes . Addition of human urokinase to cultures of mucoid or nonmucoid variants of Pseudomonas aeruginosa (strain PAO and clinical isolates from patients with cystic fibrosis) or Pseudomonas cepacia incubated in a minimal medium under nonshaking (oxygen limited) conditions led to dose-dependent enhancement of bacterial growth . The enzyme exhibited a minimal effect on the growth of bacteria when cultured under more intense aeration conditions . This enhancement of bacterial growth by urokinase required the presence of active enzyme and was not detected with inactivated enzyme or noncatalytic domains of the enzyme . Enhancement of bacterial growth was not observed following incubation of P . aeruginosa with other proteinases including thrombin, neutrophil elastase, trypsin, chymotrypsin, or pseudomonas elastase and pseudomonas alkaline proteinase . Therefore, the observed effect of urokinase was relatively specific for this enzyme . As urokinase is a natural constituent of the lung, this enzyme could contribute to bacterial growth during pulmonary infections, particularly in an inflammatory environment in which the oxygen tension may be reduced. Protein Eng, 1994 Apr, 7(4), 523 - 9 The structure-function relationship of the lipases from Pseudomonas aeruginosa and Bacillus subtilis; Misset O et al.; Within the BRIDGE T-project on lipases we investigate the structure-function relationships of the lipases from Bacillus subtilis and Pseudomonas aeruginosa . Construction of an overproducing Bacillus strain allowed the purification of > 100 mg lipase from 30 l culture supernatant . After testing a large variety of crystallization conditions, the Bacillus lipase gave crystals of reasonable quality in PEG-4000 (38-45%), Na2SO4 and octyl-beta-glucoside at 22 degrees C, pH 9.0 . A 2.5 A dataset has been obtained which is complete from 15 to 2.5 A resolution . P.aeruginosa wild-type strain PAC1R was fermented using conditions of maximum lipase production . More than 90% of the lipase was cell bound and could be solubilized by treatment of the cells with Triton X-100 . This permitted the purification of approximately 50 mg lipase . So far, no crystals of sufficient quality were obtained . Comparison of the model we built for the Pseudomonas lipase, on the basis of sequences and structures of various hydrolases which were found to possess a common folding pattern (alpha/beta hydrolase fold), with the X-ray structure of the P.glumae lipase revealed that it is possible to correctly build the structure of the core of a protein even in the absence of obvious sequence homology with a protein of known 3-D structure. Kansenshogaku Zasshi, 1994 Apr, 68(4), 500 - 7 {Alteration of serotype and drug susceptibility of some Pseudomonas aeruginosa isolates by exposure to human serum and polymorphonuclear leukocytes}; Kobayashi I et al.; In the presence of 40% human serum plus polymorphonuclear leukocytes (PMNs, 10(7) cells/ml), changes in the surface properties of clinical isolates of P . aeruginosa were investigated by determining their serotypes and pyocin types as the markers . Furthermore, three isolates were tested for their susceptibility to anti-pseudomonal drugs and profiles of outer membrane proteins by the SDS-PAGE analysis . P . aeruginosa No . 21 which did not change in serotype and pyocin type after exposure to serum plus PMNs did not alter their susceptibility to all the drugs tested or their profiles of OMPs . In the case of P . aeruginosa No . 1-S, the variants with different serotypes were formed after the exposure, and increased their susceptibilities to some beta-lactams and norfloxacin which could penetrate into the bacterial cells through the porin channels of the outer membrane . Furthermore, two of the three type variants formed decreased their susceptibilities to gentamicin and polymyxin B which penetrated into the cells by the self-promoted uptake pathway . P . aeruginosa No . 1-R formed the serotype A and G variants with different pyocin types, only when exposed to serum plus PMNs for 24 hours, and the results were accompanied by the appearance of the porin D2 which was not detected in the parent cells . A small number of P . aeruginosa formed the variants with different serotypes and pyocin types owing to the alteration of their surface structures, when the cells were exposed to serum plus PMNs . The alterations were accompanied by the changes in some outer membrane proteins and the drug susceptibility to anti-pseudomonal drugs.(ABSTRACT TRUNCATED AT 250 WORDS) Kansenshogaku Zasshi, 1994 Apr, 68(4), 479 - 85 {A clinical study of Pseudomonas pneumonia diagnosed by transtracheal aspiration}; Maeda K et al.; We performed a clinical study of 16 cases (18 episodes) of Pseudomonas pneumonia by transtracheal aspiration (TTA) from April 1983 to March 1993 . The isolation rate of Pseudomonas aeruginosa (P . aeruginosa) among 235 episodes of pneumonias with positive microorganism by TTA was 7.7% . All patients had one or more underlying disease . The most frequent underlying disease was chronic lower respiratory tract infection, followed by lung epidermoid cell carcinoma . More than half of the cases had been given antibiotics prior to the occurrence of pneumonia, and administration of adrenal corticosteroid, heavy smoking habit, and aspiration were seen as other predisposing factors concerning the onset of pneumonias . Monomicrobial infection of P . aeruginosa was 61.1%, and polymicrobial infection containing P . aeruginosa was 38.9% . Community-acquired pneumonia was 61.1% and hospital-acquired pneumonia was 38.9%; the rates of polymicrobial infections and prior administration of antibiotics were higher in the latter group . Mortality due to Pseudomonas pneumonia was 27.8% and the levels of serum albumin and total protein at the onset of pneumonia were significantly lower in the fatal cases than in the recovered cases . It was considered that not only general underlying disease which weaken the immunological resistance of the host, but local bronchial lesion was also important to the onset of the Pseudomonas pneumonia . Also, nutrition is an important prognostic factor on the host's side. J Clin Microbiol, 1994 Apr, 32(4), 1027 - 30 Accuracy and cost of antibiotic susceptibility testing of mixed morphotypes of Pseudomonas aeruginosa; Morlin GL et al.; Chronic Pseudomonas aeruginosa colonization of the lower respiratory tract of patients with cystic fibrosis frequently results in pulmonary exacerbations requiring treatment with antimicrobial agents . Multiple morphotypes with different antibiotic susceptibilities are often isolated from a single sputum sample . Determination of MICs of antibiotics for each sputum morphotype is used to guide therapy but is time-consuming and expensive . We explored an alternative assay for determining MICs for all P . aeruginosa morphotypes cultured from a homogenized sputum sample . We sought correlations of those MICs with the MIC for the most resistant morphotypes tested separately . The MICs determined for a mixture of morphotypes correctly predicted the highest MICs (+/- one dilution) determined for isolated morphotypes 73.5% of the time . The MIC for the mixed morphotypes correctly predicted susceptibility in 90.4% of samples . In contrast, determination of the MIC for the mixture of morphotypes correctly predicted resistance in only 57.0% . For sputa containing susceptible isolates, testing the mixed culture may provide adequate susceptibility data with significant laboratory time and cost savings . However, for sputa with resistant strains, the traditional method of testing isolated morphotypes should still be used. Tissue Cell, 1994 Apr, 26(2), 181 - 8 The effect of anti-exotoxin A on the adherence of Pseudomonas aeruginosa to hamster tracheal epithelial cells in vitro; Moller PC et al.; One of the most important initial events of colonization and infection of epithelial tissues is the adherence of bacteria to mucosal surfaces . Bacterial adhesion to the epithelial cell may be mediated by a variety of adhesins, including exoproducts . One of these exoproducts, exotoxin A (EA) is a three-domain bacterial toxin that kills mammalian cells by gaining entry to the cytosol and inactivating protein synthesis . In the present study, HTE cultures, 2-4 weeks in vitro (containing both ciliated and non-ciliated cells), were treated for 1 hr with two different non-mucoid strains of Pseudomonas aeruginosa (1 x 10(8) organisms/ml) in the presence of anti-EA . 50 randomly selected fields were evaluated via SEM at x2500 magnification and the number of bacterial clusters/field quantitated . The results of this study indicate, first, that both piliated (ATCC15692) and non-piliated (PAKp) P . aeruginosa will bind to the HTE cells and, second, that treatment of HTE cells with either strain of P . aeruginosa in the presence of anti-EA will reduce bacterial binding by 25% to 50% . Thus, EA may participate in the adhesion of P . aeruginosa to respiratory tract epithelia. Mol Ecol, 1994 Apr, 3(2), 121 - 6 Transduction of a freshwater microbial community by a new Pseudomonas aeruginosa generalized transducing phage, UT1; Ripp S et al.; A pseudolysogenic, generalized transducing bacteriophage, UT1, isolated from a natural freshwater habitat, is capable of mediating the transfer of both chromosomal and plasmid DNA between strains of Pseudomonas aeruginosa . Several chromosomal alleles from three different P . aeruginosa strains were found to transduce at frequencies from 10(-8) to 10(-10) transductants per PFU at multiplicities of infection (MOI) between 0.1 and 1 . Transduction frequencies of certain alleles increased up to 1000-fold as MOIs were decreased to 0.01 . UT1 is also capable of transducing plasmid DNA to indigenous populations of microorganisms in natural lake-water environments . Data obtained in this study suggest that environmentally endemic bacteriophages such as UT1 are formidable transducers of naturally occurring microbial communities . It should be possible to develop model systems to test transduction in freshwater environments using components derived exclusively from these environments. Vaccine, 1994 Apr, 12(5), 457 - 64 Antigenic competition in a multivalent foot rot vaccine; Hunt JD et al.; The antigenic competition that occurs when pilus antigens of different serogroups are combined in multivalent vaccines for foot rot has been investigated using recombinant pilus antigens . Our prototype vaccine contains pili from nine serogroups of Dichelobacter nodosus which are expressed in Pseudomonas aeruginosa . Sheep inoculated with this multivalent vaccine were not as well protected against foot rot as those given the monovalent vaccine . Levels of agglutinating and total antibody specific for any particular pili serogroup were found to be significantly reduced in sheep vaccinated with six or more closely related pili . This effect was more pronounced for agglutinating antibody, which is thought to mediate protection, but was also observed with total antibody levels measured by ELISA . The antigenic competition was not associated with the total antigen load as a tenfold higher dose of monovalent pili induced high titres of antibody . Furthermore, distributing the vaccine to four sites, each draining to a different lymph node, failed to overcome the competition . Experiments with mixtures of monospecific sera indicate that the phenomenon is unlikely to be due to blocking of serogroup-specific protective antibodies by an excess of cross-reactive non-protective antibody elicited by heterologous pili. Pathol Biol (Paris), 1994 Apr, 42(4), 293 - 5 Susceptibility of multiresistant serotype 012 Pseudomonas aeruginosa to fosfomycin in combination with other antibiotics; Watine J et al.; Serotype 012 represented 15% of 244 isolates of Pseudomonas aeruginosa isolated in the hospital over a 2 year-period and most isolates of this serotype were resistant to multiple antibiotics . Combination experiments showed that fosfomycin and amikacin together were active against 92% of 012 isolates . It is recommended that serotyping be used systematically to identify 012 strains rapidly and fosfomycin/amikacin be considered as a presumptive antipseudomonal therapy in 012 infections. Biochim Biophys Acta, 1994 Mar 16, 1205(1), 139 - 45 Arg-188 and Trp-144 are implicated in the binding of urea and acetamide to the active site of the amidase from Pseudomonas aeruginosa; Tata R et al.; Urea is a time-dependent active-site-directed inhibitor of Pseudomonas aeruginosa amidase . We found that 20 mM hydroxylamine caused bound urea to be released from the inactive urea:amidase complex with the restoration of enzyme activity . Bound urea restricts the titrability of the enzyme's -SH groups to 6 per hexameric molecule and protects it against thermal denaturation suggesting that urea binding provokes a conformational change in the enzyme . Mutations in the P . aeruginosa amidase gene that reduce the binding affinity of the enzyme for both urea and the substrate acetamide have been identified by direct sequencing of PCR-amplified mutant genes and confirmed by sequencing cloned PCR-amplified genes . The mutations were in two regions of the enzyme substituting either Arg-188 (or Gln-190, in one case) or Trp-144; one amidase that bound neither urea nor acetamide was doubly mutant with an amino-acid change at both sites. Arch Intern Med, 1994 Mar 14, 154(5), 586 - 9 Ceftazidime-related nonconvulsive status epilepticus; Klion AD et al.; The third-generation cephalosporin, ceftazidime, is widely used for the treatment of serious gram-negative infections . As is true of cephalosporins in general, reported adverse effects have been few . We report a case of ceftazidime-induced status epilepticus in a patient with Pseudomonas aeruginosa meningitis and compare the clinical manifestations of this case with those of two previously described cases of ceftazidime-related encephalopathy . This diagnosis should be entertained and an electroencephalogram should be obtained in all patients with myoclonus and/or altered mental status while they are receiving ceftazidime therapy. Gene, 1994 Mar 11, 140(1), 7 - 15 Utilization of a mini-Dlac transposable element to create an alpha-complementation and regulated expression system for cloning in Pseudomonas aeruginosa; Karkhoff-Schweizer RR et al.; A lac-based alpha-complementation and expression system was developed for use in molecular cloning in Pseudomonas aeruginosa . A bacteriophage D3112-based mini-Dlac transposable element, containing the lacIq-regulated lacZ delta M15 gene next to a selectable marker, was constructed . Mixed D3112 lysates were used to transduce P . aeruginosa PAO1, and derivatives containing randomly inserted chromosomal copies of the mini-Dlac element were obtained . Transformation of the PAO1::mini-Dlac transductants with the broad-host-range vector, pUCP19, led to the formation of blue colonies on indicator medium in the presence of inducer . In contrast, transformants harboring the pUCP19 derivative pCDO, containing the catechol-2,3-dioxygenase (C23O)-encoding xylE gene under lac promoter control, were white on the same medium . Expression of xylE was tightly controlled by single-copy mini-Dlac-encoded lac repressor and in induced cultures was increased more than 100-fold over that observed in uninduced cultures . The usefulness of the system for molecular cloning in P . aeruginosa was demonstrated by ligating size-fractionated PAO1 chromosomal fragments into pUCP19, followed by transformation of the newly isolated PAO1::mini-Dlac host . All randomly chosen white colonies contained recombinant plasmids, with inserts of the correct size range, while blue colonies contained pUCP19 alone . The functionality of the system was also shown in another frequently studied strain, PA103. Schweiz Med Wochenschr, 1994 Mar 5, 124(9), 362 - 7 {Fire eater's lung (hydrocarbon pneumonitis)}; Borer H et al.; Hydrocarbon pneumonitis is usually related to accidental poisoning in children . We describe a case of hydrocarbon pneumonitis after petroleum aspiration in an adult fire eater . The main symptoms were cough, dyspnea, chest pain and fever . The patient showed bilateral infiltrates in the middle and lower parts of the lung, hypoxemia and a restrictive ventilatory defect . The evolution was complicated by superinfection with Pseudomonas aeruginosa and by pneumatoceles . The acute stage lasted three weeks, and the patient recovered without sequelae within two months . Prophylactic application of antibiotics and corticosteroids cannot be recommended for prevention of hydrocarbon pneumonitis. Respir Med, 1994 Mar, 88(3), 203 - 11 The quantitative distribution of nebulized antibiotic in the lung in cystic fibrosis; Mukhopadhyay S et al.; Nebulized antibiotic therapy in cystic fibrosis is an established procedure . The present study was designed to quantitate deposition, and assess its relation to the disease state . Twenty seven children and young adults with cystic fibrosis (mean 11.6 years, range 4-23 years, 12 females) were studied to establish the quantity and pattern of deposition of nebulized tobramycin in the respiratory tract . A single (120 mg) dose of nebulized 99 m technetium-labelled tobramycin was administered, and imaged with a gamma-camera . The mean penetration index (which compares the distribution of 81 m-Krypton gas with Tc-radioaerosol) was also used to measure peripheral deposition efficiency . The aerosol mass median diameter (MMAD) for the compressor-nebulizer system used was 5.3 u, measured with the Malvern Mastersizer . Serial sputum samples were fluroimmunoassayed for tobramycin in nine patients . A mean of 8.0 (SEM 1.0) mg tobramycin reached the lungs . There was no relationship between the total pulmonary deposition and indices of pulmonary damage in cystic fibrosis . Sixteen percent of the lung tobramycin reached the periphery . The greater the lung damage as indicated by FEV1 and Chrispin-Norman scores, the smaller the proportion of pulmonary tobramycin that reached the periphery . The mean penetration index increased with increase in the FRC, but bore no relation to other respiratory function tests or to chest X-ray scores . Sputum tobramycin concentrations reached levels bactericidal for Pseudomonas aeruginosa . Airway obstruction and damage affected the proportion of pulmonary tobramycin reaching the periphery . The proportion of tobramycin reaching the lungs was small and variable.(ABSTRACT TRUNCATED AT 250 WORDS) Antimicrob Agents Chemother, 1994 Mar, 38(3), 528 - 33 Comparative in vitro exoenzyme-suppressing activities of azithromycin and other macrolide antibiotics against Pseudomonas aeruginosa; Mizukane R et al.; The inhibitory effects of azithromycin (AZM), a new 15-membered macrolide antibiotic, on the production of exotoxin A, total protease, elastase, and phospholipase C by Pseudomonas aeruginosa were determined, and the virulence-suppressing effects of AZM were compared with those of erythromycin (EM), roxithromycin (RXM), and rokitamycin (RKM) . The effect of exposure of P . aeruginosa PA103 or B16 in cultures to sub-MICs of these macrolide antibiotics on the production of exoenzymes was determined . AZM suppressed the in vitro production of extracellular and intracellular exotoxin A by P . aeruginosa PA103 more than did EM, even at a concentration of only 2 micrograms/ml . At concentrations of between 4 and 32 micrograms/ml, AZM also inhibited total protease, elastase, and phospholipase C production by P . aeruginosa B16 more than did EM, RXM, and RKM . AZM was effective in suppressing exotoxin A and total protease production through 24 h of incubation in the presence of drug at sub-MICs, but it had no significant effect on either the growth of P . aeruginosa or its total protein production . Moreover, at a concentration of 4 micrograms/ml, AZM suppressed exoenzyme production by other strains of P . aeruginosa more than did EM . These findings indicate that AZM, EM, RXM, and RKM each has an inhibitory effect on exoenzyme production separate from the antimicrobial effect and that, of these macrolides, AZM has the strongest virulence-suppressing effect. J Am Optom Assoc, 1994 Mar, 65(3), 161 - 3 A comparative study of techniques for decreasing contact lens storage case contamination; Larragoiti ND et al.; BACKGROUND: Contact lens storage cases may harbor a variety of pathogenic organisms, and are a potential source of ocular infection . In this study, we evaluated the anti-microbial efficacy of several methods of rinsing of contact lens storage cases . METHODS: Lens cases were inoculated with Pseudomonas aeruginosa and then 1) rinsed with hot tap water and closed without drying, 2) rinsed with hot tap water followed by air drying, 3) rinsed with 3% hydrogen peroxide followed by air drying, or 4) left closed and undisturbed (control condition) . After 24 hours, the cases were cultured to determine the rate of disinfection . RESULTS: The hydrogen peroxide rinse was the most effective (99.5 percent of cases disinfected), followed by hot water with air drying (94.7 percent) and hot water without drying (51.1 percent) . Significant residual hydrogen peroxide was detected in the cases rinsed with peroxide . CONCLUSIONS: Therefore, we recommend that patients be instructed to rinse their cases with hot water and allow them to air dry after use, as well as replace their cases on a regular basis. J Clin Microbiol, 1994 Mar, 32(3), 666 - 71 Pseudomonas aeruginosa serotype O12 outbreak studied by arbitrary primer PCR; Elaichouni A et al.; A total of 16 colonizing and infecting ofloxacin-resistant Pseudomonas aeruginosa strains and two strains isolated from ventilation equipment fluids, all with similar colonial morphologies and with minor but distinct susceptibility differences, were suspected of belonging to a single outbreak and were studied by arbitrary primer (AP) PCR . Thirteen nonrelated strains were included to evaluate the discriminatory capacity of the technique . AP PCR fingerprinting was compared with serotyping, phage typing, and antibiotic susceptibility testing . AP PCR was performed independently with three different primers . The different AP PCR typing systems yielded almost identical patterns for the epidemic strains and enabled us to differentiate most of the nonrelated strains from each other and from the outbreak strains . The combination of AP PCR typing and the phenotyping techniques that we used enabled us to conclude that an outbreak was occurring . In general, the typeability of AP PCR was greater than those of phage typing and serotyping, while the discriminatory powers of the three methods were comparable. Eur J Pediatr, 1994 Mar, 153(3), 158 - 63 Perspectives of longitudinal growth in cystic fibrosis from birth to adult age; Haeusler G et al.; The longitudinal growth in 139 patients with cystic fibrosis (CF) was investigated from birth until the age of 19 years . Already at birth weight and length were reduced (weight: -0.83 +/- 0.13 SDS in girls, -0.44 +/- 0.13 SDS in boys; length: 0.55 +/- 0.13 SDS in girls, -0.39 +/- 0.14 SDS in boys; mean +/- SEM) . Both variables showed a further decline until diagnosis was established (weight: -1.57 +/- 0.21 SDS in girls, -1.46 +/- 0.25 SDS in boys; length: -1.15 +/- 0.32 SDS in girls, -1.03 +/- 0.52 SDS in boys; mean +/- SEM) . Six to 12 months after diagnosis length improved and reached the 25th percentile in both sexes . Height and weight followed the 25th percentile throughout childhood . Growth velocity was fairly normal during this period . There was a loss in percentiles of both height and weight after the age of 8 years and the pubertal growth spurt was delayed and reduced . However, the 25th percentile was reached again in the adolescent period . At the age of 19 years median height was 161.5 cm in girls and 173 cm in boys, both representing the 25th percentile . Using a sensitive statistical method for analysis of growth data we present CF specific growth curves for height, weight and growth velocity . There was no significant effect of pulmonary colonization with Pseudomonas aeruginosa on growth velocity. Nippon Geka Gakkai Zasshi, 1994 Mar, 95(3), 141 - 8 {A study of nosocomial infections by the comparison of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa}; Takesue Y et al.; The objective of this study was to investigate nosocomial infections by the comparison of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa . We made a serological classification of 262 strains of Pseudomonas aeruginosa between 1983 and 1991 . Group E strains were prevalent in 1987 and group F strains after 1990 . Both these groups strains were resistant to several antibiotics, and were scarcely detected from the appendix contents in appendicitis that originated outside the hospital . Therefore we presumed these were nosocomial pathogens . With the countermeasures to nosocomial infections from 1987 on the isolation rate of enterotoxin type B and C MRSA strains decreased . Following this MRSA change, almost group E strains disappeared in Pseudomonas aeruginosa . Whereas type AC MRSA began to isolated abruptly from 1990, and the isolation of group F strains increased simultaneously . Type B and C strains still remained high sensitivity to minocycline . Type AC which have already gotten resistance to minocycline took the place of type B and C and prevalented . Similarly group F in Pseudomonas aeruginosa took the place of group E as soon as the former acquired resistance to carbapenem. Kansenshogaku Zasshi, 1994 Mar, 68(3), 314 - 8 {The inhibitory effect of mouse monoclonal IgM antibody E5 against endotoxin on limulus activity of various lipopolysaccharides}; Kobayashi H et al.; The anti-endotoxin activity of mouse monoclonal IgM antibody E5 was determined against various lipopolysaccharides (LPSs) derived from Escherichia coli J5, E . coli O111:B4, Klebsiella pneumoniae and Pseudomonas aeruginosa by measuring limulus activity by endospecy method . In general, above 0.2 microgram/ml of E5 reduced almost dose-dependently the limulus activities of all LPSs tested, although the degree of reduction in limulus activity somewhat varied among bacterial species . The limulus activities of LPS from E . coli J5, E . coli O111:B4, K . pneumoniae and P . aeruginosa decreased by 72%, 50%, 44%, and 58%, respectively, by exposure to 5.0 micrograms/ml of E5 for 2 hrs. Pediatr Dermatol, 1994 Mar, 11(1), 35 - 8 Recurrent Pseudomonas folliculitis; Trueb RM et al.; Pseudomonas aeruginosa has attracted the attention of the dermatologic literature through repeatedly reported outbreaks of folliculitis occurring in small epidemics related to contaminated whirlpools, hot tubs, and swimming pools . In contrast, sporadic cases without these recreational exposures may present a diagnostic challenge . Two sisters, age 8 and 4 years, had sporadic Pseudomonas folliculitis . It is important to recognize the organism as the cause of this recurrent, papulopustular, follicular eruption on the trunk and gluteal regions, also called nonrecreational Pseudomonas folliculitis, to avoid misdiagnosis and mismanagment . Inadvertent therapy with steroids may result in rapid spread of the lesions . Unless a meticulous investigation of contaminant sources in the environment leads to the detection of the vehicle of infection, the dermatitis tends to recur and become chronic. Biochem J, 1994 Mar 1, 298 ( Pt 2), 329 - 34 Electron transfer from Phanerochaete chrysosporium cellobiose oxidase to equine cytochrome c and Pseudomonas aeruginosa cytochrome c-551; Rogers MS et al.; The electron-transfer reactions of cellobiose oxidase (CBO) have been investigated by conventional and by rapid-scan stopped-flow spectroscopy at pH 6.0 . Analysis of the absorbance/time/wavelength matrix by Singular Value Decomposition (SVD) confirms earlier studies showing that cellobiose rapidly reduces the flavin group (7.7 s-1; cellobiose, 100 microM) which in turn slowly (0.2 s-1) reduces the cytochrome b moiety . In the presence of CBO, cellobiose reduces cytochromes c in a reaction that does not depend on oxygen or superoxide . The rate limit for this process is independent of the source of the cytochromes c and is identical with the rate of cytochrome b reduction . Rapid-mixing experiments show that cytochrome b may donate electrons very rapidly to either mammalian cytochrome c or bacterial cytochrome c-551 . The reactions were second-order (kc = 1.75 x 10(7) M-1 x s-1; kc-551 = 4.3 x 10(6) M-1 x s-1; pH 6.0, 21 degrees C and I0.064) and strongly ionic-strength (I)-dependent: kc decreasing with I and kc-551 increasing with I . These results suggest the electron-transfer site near cytochrome b bears a significant negative charge . Equilibrium gel chromatography confirms that CBO oxidase and positively charged mammalian cytochrome c make stable complexes . These results are discussed in terms of a model suggesting an electron-transfer role for cytochrome b in vivo, possibly connected with radical-mediated cellulose breakdown. Arch Surg, 1994 Mar, 129(3), 325 - 8 Additive effects of thermal injury and infection on gut permeability; Ryan CM et al.; OBJECTIVE: To determine the effects of burn size and burn wound infection on gut permeability to the macromolecule polyethylene glycol 3350 (PEG 3350; molecular weight, 3350 d) . DESIGN: Randomized, controlled study using 36 male Sprague-Dawley rats . SETTING: Hospital research laboratory . INTERVENTIONS: Animals received scald burns to 0%, 20%, or 35% of their total body surface area . Half of the animals in each group were infected with Pseudomonas aeruginosa . MAIN OUTCOME MEASURES: Gut permeability was measured using the intestinal absorption and renal excretion of enterally administered PEG 3350 and mannitol (molecular weight, 182 d) . RESULTS: There were dramatic increases in PEG 3350 excretion and in the PEG 3350/mannitol ratios (P = .0001 in both instances) that were seen in relation to burn size . Significant increases in PEG 3350 excretion and in the PEG 3350/mannitol ratios (P = .017 and P = .045, respectively) were also seen in animals in which infection was present . This was in addition to the effects of burn size already noted . CONCLUSIONS: A direct relationship between gut permeability and the extent of burn injury was found, which is consistent with the results from a previous study in humans . In addition, this study found that further separate increases in gut permeability occur in the presence of P aeruginosa in burn wound infections. Am J Respir Crit Care Med, 1994 Mar, 149(3 Pt 1), 687 - 93 Extracellular metabolites of Pseudomonas aeruginosa produce bronchoconstriction by different mechanisms; Forteza R et al.; Bacterial supernatants (BS) obtained from broth cultures of Pseudomonas aeruginosa cause bronchoconstriction in sheep, suggesting that BS contain proinflammatory metabolites . In this study we investigated the mechanism(s) responsible for this bronchial effect . BS were obtained from 48 h cultures and sterilized by filtration . Sheep (n = 6) were intubated and swallowed an esophageal balloon for the measurement of specific lung resistance (SRL) . Aerosols of BS (3 ml total) immediately increased SRL (541%) . Neither aerosolized broth (control) nor inhaled endotoxin in excess of that contained in the BS had an effect . BS challenges were repeated on separate occasions except that the sheep were treated 30 min before challenge with the anticholinergic agent atropine (0.2 mg/kg, intravenously); the anti-allergic agent nedocromil (1 mg/kg, aerosol); the histamine H1 antagonist chlorpheniramine (2 mg/kg); or the bradykinin (BK) B2 receptor antagonists NPC-567 (5 mg/ml, aerosol) or NPC-17761 (1 mg/ml aerosol) . The results showed that greater than 90% protection (p < 0.05) was achieved when the animals were pretreated with atropine, nedocromil sodium, or either of the two BK antagonists, but only 27 +/- 21% protection was seen with chlorpheniramine pretreatment . These findings are characteristic of a BK-mediated response . Analysis of bronchoalveolar lavage fluid obtained before and after BS challenge confirmed that i-kinins, but not histamine, increased (p < 0.05) from 61 +/- 7 to 304 +/- 55 pg/ml . Control (broth) challenges produced no such change . To identify the metabolites involved, we tested the effects of aerosolizing two suspected components of BS, 1-hydroxyphenazine (1-HP) and pyocyanine (PYO) in five sheep.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1994 Mar, 176(5), 1316 - 22 Nucleotide sequence of the rpoN gene and characterization of two downstream open reading frames in Pseudomonas aeruginosa; Jin S et al.; The rpoN gene of Pseudomonas aeruginosa is required for the expression of a number of diverse genes, ranging from several classes of bacterial adhesins to enzymes for amino acid biosynthesis . The nucleotide sequence of the rpoN gene and its flanking region has been determined . The deduced amino acid sequence of the rpoN product is highly homologous to sequences of RpoN proteins of other microorganisms . Moreover, two open reading frames (ORF1 and ORF2) encoding peptides of 103 and 154 amino acids long, respectively, were found downstream of the rpoN gene . These two ORF products have a high degree of amino acid sequence homology with products of similar ORFs located adjacent to the rpoN genes in other microorganisms . Mutations in either ORF lead to a significant increase in P . aeruginosa generation time when propagated on minimal medium . These mutations had no effect on the expression of pilin or flagellin genes, whose expression depends on RpoN . Complementation analysis showed that the two ORFs are in the same transcriptional unit and the growth defects of the two ORF mutants on minimal medium are due to mutational effects on ORF2 . The adverse effect of the ORF mutations on the growth of P . aeruginosa in minimal media can be suppressed by the addition of glutamine but not arginine, glutamate, histidine, or proline . Since rpoN mutants of P . aeruginosa display this same amino acid requirement for growth, the ORF2 product very likely functions as a coinducer of some but not all of the RpoN-controlled genes. Infect Immun, 1994 Mar, 62(3), 897 - 903 Characterization of Pseudomonas aeruginosa mutants that are deficient in exotoxin A synthesis and are altered in expression of regA, a positive regulator of exotoxin A; West SE et al.; In Pseudomonas aeruginosa, production of exotoxin A, an ADP-ribosyltransferase, is a complex and highly regulated process . Two positively acting regulatory genes, regA and regB, have been cloned and characterized . To identify additional exotoxin A regulatory genes, we have characterized four N-methyl-N'-nitro-N-nitrosoguanidine-generated mutants of P . aeruginosa PA103 which are deficient in exotoxin A production . These mutants (PA103-8, PA103-15, PA103-16, and PA103-19) do not accumulate intracellular exotoxin A and are not complemented by the cloned toxA or regAB genes . This observation indicates that the lesion(s) in the mutants is probably in an exotoxin A regulatory gene(s) and is not in the genes for secretion of exotoxin A or in the toxA or regAB genes . To assess the effect of the putative regulatory mutations on the toxA and regAB genes, we compared the activity of the toxA and regAB promoters in the mutant and parental strains using plasmids containing the genes for beta-galactosidase or chloramphenicol acetyltransferase under the control of either the toxA or the regAB promoter . The toxA promoter-beta-galactosidase fusion plasmid could not be maintained in PA103-8 . beta-Galactosidase expression driven by the toxA promoter was absent in the mutant PA103-19 and occurred at a low level, which was not repressed by iron in mutants PA103-15 and PA103-16 . The regAB genes are temporally controlled by two promoters, P1 and P2 . In all four mutants, regAB P1 promoter activity was reduced; however, expression under the control of the regAB P2 promoter was normal . These observations suggest the existence of one or more regulatory genes which directly affect expression of both the toxA and the regAB P1 promoters. Infect Immun, 1994 Mar, 62(3), 809 - 17 Characterization of lipopolysaccharide-deficient mutants of Pseudomonas aeruginosa derived from serotypes O3, O5, and O6; Dasgupta T et al.; Well-characterized rough mutants are important for the understanding of structures, functions, and biosynthesis of lipopolysaccharide (LPS) in gram-negative organisms . In this study, three series of Pseudomonas aeruginosa LPS-deficient mutants, namely PAC strains derived from serotype O3, AK strains derived from strain PAO1 (serotype O5), and serotype O6-derived mutants were subjected to biochemical analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining as well as immunochemical characterization using LPS-specific monoclonal antibodies . The O-side-chain deficiency among the O6-derived mutants was also examined, and three mutants, A28, R5, and H4, were subsequently chosen for the elucidation of component sugars of the core structure of serotype O6 LPS . LPS of strain A28 has L-rhamnose and proportionally higher amounts of D-glucose, a feature shared by the O5-derived mutant, strain AK1401 (previously demonstrated as a mutant with a core-plus-one O repeat) . In contrast strains R5 and H4 were shown to be devoid of L-rhamnose and have low and undetectable amounts of D-glucose, respectively, which indicated their core deficiency . The LPS-deficient or -sufficient characteristics of the P . aeruginosa strains examined correlated will with serum sensitivity data . This report represents a comprehensive analysis of rough mutants derived from O3 and O5 strains that have been used by others in many studies and a first look at the core oligosaccharide region of serotype O6 LPS obtained with the O6-derived mutants generated in this study. J Biomed Mater Res, 1994 Mar, 28(3), 303 - 9 Poloxamer 407 as a bacterial adhesive for hydrogel contact lenses; Portoles M et al.; Bacterial adherence and colonization of biomedical prosthetic implants is one of the main causes for implant withdrawal . The abhesive (anti-adhesive) effect of poloxamer 407 on several Gram-positive and Gram-negative strains and the site of action of its effect have been investigated . Poloxamer 407 significantly inhibited 92-99% of Pseudomonas aeruginosa adherence to hydrophilic contact lenses (P < .05) . This adherence inhibition was concentration-dependent . A reduction of about 50-60% was obtained for Staphylococcus strains, and 50-70% for Gram-negative strains other than Pseudomonas . The poloxamer seems to act on the bacteria surface, but not on the contact lens surface . Poloxamer 407 could potentially prevent implant-related infections and keratitis associated with contact lens wear, by inhibiting bacterial adherence onto the implant or contact lens surface. Clin Ther, 1994 Mar-Apr, 16(2), 236 - 52 Combined aztreonam and gentamicin therapy for pseudomonal lower respiratory tract infections; Andrews R et al.; A multicenter, open-label study was performed to assess the efficacy and safety of aztreonam plus gentamicin in the treatment of lower respiratory tract infections due to Pseudomonas aeruginosa . Patients with documented P aeruginosa infections were given aztreonam 2 g every 8 hours (q8h) plus gentamicin 3 to 5 mg/kg per day in three equal doses . Clindamycin, 600 mg q8h, was added to the regimen for patients with infections also involving gram-positive and/or anaerobic bacteria . Therapy was continued for at least 5 days or until obvious failure to respond to treatment . Of 64 patients with suspected P aeruginosa infections, 57 were eligible for clinical evaluation and 51 for microbiologic evaluation . At entry, impaired host defense was present in 35% of patients, and chronic obstructive pulmonary disease in 28%, in addition to other predisposing conditions such as emphysema, history of tuberculosis, and pneumothorax . The clinical response rate for the combination regimen was 48/57 (84%), which included 27 (47%) cures and 21 (37%) partial responses . The microbiologic response rate was 35/51 (69%), of which 25 (49%) outcomes were classified as eradication and 10 (20%) as eradication with relapse . Superinfection was observed in 3 (6%) patients . The combination of aztreonam and gentamicin was synergistic in the initial isolates obtained from 33 (72%) patients . A total of 16 patients died of pulmonary or other underlying disease, for a mortality rate of 28% . The monobactam-aminoglycoside combination was generally well tolerated . Two other patients were withdrawn because rashes emerged on treatment . This study demonstrates that aztreonam can be administered as one component of a synergistic monobactam-aminoglycoside therapy in the treatment of nosocomial lower respiratory tract infections involving P aeruginosa. J Biochem (Tokyo), 1994 Mar, 115(3), 532 - 5 Inhibition of Streptomyces griseus metallo-endopeptidase II (SGMPII) by active-site-directed inhibitors; Kumazaki T et al.; Inactivation of Streptomyces griseus metallo-endopeptidase II (SGMPII) by ClCH2CO-DL-(N-OH)Leu-OCH3 and by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 was studied kinetically . These reagents cause irreversible inhibition of the enzyme in a pseudo-first order reaction, and the inhibition reaction exhibits saturation kinetics . The second-order rate constants for inactivation of SGMPII by ClCH2CO-DL-(N-OH)Leu-OCH3 and by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 were measured to be 0.12 and 8.9 M-1.s-1, respectively . The order of affinities of metallo-endopeptidases towards these irreversible inhibitors is thermolysin > SGMPII > Pseudomonas aeruginosa elastase . A competitive inhibitor of SGMPII, L-Val-L-Trp, protects the enzyme against inactivation by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 in a competitive manner . Furthermore, the pH profile of the inactivation closely resembles that for the hydrolysis of synthetic peptide substrates by the enzyme . These findings suggest that these reagents bind reversibly and react irreversibly at the active site of the enzyme. J Antimicrob Chemother, 1994 Mar, 33(3), 563 - 9 Conditions for the emergence of resistance to cefpirome and ceftazidime in experimental endocarditis due to Pseudomonas aeruginosa; Fantin B et al.; The conditions for the emergence of resistance to cefpirome and ceftazidime were studied in rabbits with experimental aortic endocarditis due to Pseudomonas aeruginosa . The MIC of cefpirome was 16 mg/L and that of ceftazidime was 4 mg/L . Resistant mutants with MICs of > or = 64 mg/L were obtained in vitro to cefpirome after a single passage and to ceftazidime after five passages . A single dose of 50 mg/kg intramuscularly gave mean peak serum concentrations of 110.0 +/- 31.7 mg/L for cefpirome compared with 67.7 +/- 21.4 mg/L for ceftazidime and the half-lives were 1.2 +/- 0.1 h and 2.1 +/- 0.4 h, respectively . After treating infected rabbits for 4 days with various dosing regimens, resistant strains were only detected in those animals in which the time that the serum concentration exceeded the MIC was less than half of the dosing interval . There was no evidence of emergent resistance when the serum concentrations exceeded the MIC for a longer period nor when amikican was combined with the cephalosporins on the first day of therapy . Moreover, once differences in MICs and pharmacokinetics were taken into account, both antibiotics had a similar propensity to induce resistance. Plasmid, 1994 Mar, 31(2), 196 - 200 Functional features of oriV of the broad host range plasmid RSF1010 in Pseudomonas aeruginosa; Higashi A et al.; The broad host range plasmid RSF1010 requires for its replication in Escherichia coli three plasmid-encoded proteins and specific nucleotide sequences ssiA, ssiB, and iterons in the oriVRSF1010 . In Pseudomonas aeruginosa, a recombinant mini-RSF1010 plasmid lacking ssiB lost its replication ability, but a miniplasmid lacking ssiA or carrying a primosome assembly site in place of ssiA could replicate . Moreover, ssiA, as a sole ssi signal, in the orientation that ssiB had originally taken was sufficient for replication of the miniplasmid . These results indicated that only one RSF1010-specific ssi signal in the orientation that ssiB takes in wild-type oriVRSF1010 was essential for replication of RSF1010 . Replication of the miniplasmids was dependent on the three plasmid-encoded proteins, RepA, B', and C, as in E . coli. Plasmid, 1994 Mar, 31(2), 148 - 57 Characterization of pVT736-1, a rolling-circle plasmid from the gram-negative bacterium Actinobacillus actinomycetemcomitans; Galli DM et al.; The presence of plasmid DNA in Actinobacillus actinomycetemcomitans has been reported . Recently, the construction of an Escherichia coli/A . actinomycetemcomitans shuttle vector, based on the A . actinomycetemcomitans-derived 2.0-kb plasmid pVT736-1, was described (D . J . LeBlanc, A . R . Abu-Al-Jaibat, P . K . Sreenivasan, and P . M . Fives-Taylor, Oral Microbiol . Immunol . (1993) 8, 94-99) . The current study was initiated with the determination of the nucleotide sequence of pVT736-1, which revealed the presence of two open reading frames (ORFs) encoding proteins of 293 (ORF1) and 95 (ORF2) amino acids . Evidence that pVT736-1 replicates via a single-stranded (ss) intermediate included: (i) the presence of ssDNA in cells and in cell-free supernatant, (ii) the presence of conserved sequence motifs in the predicted ORF1 protein that are typical of initiator (Rep) proteins associated with replication by a rolling-circle mode, and (iii) 39% amino acid identity between the putative Rep proteins of pVT736-1 and Pf3, a filamentous ssDNA bacteriophage of Pseudomonas aeruginosa . A putative plus origin of replication in pVT736-1 was located upstream of ORF1, in a 200-bp region with potential for secondary hairpin structures . The identification in gram-negative bacteria of extrachromosomal DNA (other than bacteriophage) that replicates by a rolling-circle mode and shows no extensive homology to plasmids from gram-positive organisms is rather unique. Clin Infect Dis, 1994 Mar, 18(3), 390 - 4 Pseudomonas aeruginosa bacteremia in immunocompromised children: analysis of factors associated with a poor outcome; Fergie JE et al.; We identified 98 children and adolescents with cancer who were treated for Pseudomonas aeruginosa bacteremia over a 27-year period . The most common underlying disease was leukemia (lymphoblastic in 63 cases and myeloblastic in 17); in addition, 12 episodes were associated with solid tumors and 6 with other diagnoses . There were 29.5 episodes of P . aeruginosa bacteremia/1,000 cases of acute lymphoblastic leukemia, with a mortality of 27%, and 29.8 episodes/1,000 cases of acute myeloblastic leukemia, with a mortality of 24%; patients with solid tumors had an incidence of 5.0/1,000 cases and a mortality of 58% (P = .01 for mortality in leukemia vs . mortality in solid tumors, logistic regression analysis) . Mortality was lower among children who developed bacteremia during remission therapy or induction therapy than among children who were being treated for relapse (P = .001) . The majority (78%) of the 76 evaluated cases developed during periods when the absolute neutrophil count (ANC) was < 0.1 x 10(9)/L; mortality was higher among patients with such counts than among those with higher ANCs (36% vs . 14%, P = .04) . Logistic regression analysis showed that perineal skin lesions were associated with higher mortality than were lesions at other sites of skin involvement (54% vs . 23%, P = .04). J Cell Sci, 1994 Mar, 107 ( Pt 3), 719 - 26 Genetic deficiency in low density lipoprotein receptor-related protein confers cellular resistance to Pseudomonas exotoxin A . Evidence that this protein is required for uptake and degradation of multiple ligands; Willnow TE et al.; The low density lipoprotein receptor-related protein (LRP) is a large multifunctional receptor implicated in the cellular uptake of functionally diverse ligands . Biochemical evidence suggests that LRP is a clearance receptor for apoE-rich remnant lipoproteins, lipoprotein lipase, alpha 2-macroglobulin/protease complexes, plasminogen activator/inhibitor complexes, the active protease tissue-type plasminogen activator and exotoxin A from Pseudomonas aeruginosa . Mice genetically deficient in LRP die early during gestation, underscoring the essential physiological role of this gene in vivo . To study the effect of LRP deficiency at the cellular level, we have used Pseudomonas exotoxin A (PEA) to select murine embryonic fibroblasts that are genetically deficient in LRP . Our results demonstrate that this single gene defect is sufficient to confer resistance to PEA on cultured cells . In addition, embryonic fibroblasts lacking LRP are unable to bind, internalize and degrade methylamine-activated alpha 2-macroglobulin and complexes of urokinase with plasminogen activator inhibitor-1 . Furthermore, cellular uptake and degradation of receptor-associated protein, a 39 kDa accessory protein of LRP, is reduced by 90% in the absence of LRP . These results provide genetic evidence for the multifunctional nature of LRP and its crucial role in protease/inhibitor complex metabolism. Shock, 1994 Mar, 1(3), 221 - 7 Deferoxamine induces hypotension in experimental gram-negative septicemia; Mustard RA et al.; Multiple organ system failure may result from tissue damage caused by activated neutrophils or endotoxin . A significant part of this tissue damage is due to peroxidation induced by oxygen-free radicals and requires iron as a co-factor . Iron chelation has been shown to prevent tissue damage in some models . This experiment was carried out to determine whether iron chelation with deferoxamine (DFO) would prevent lung damage in a swine model of Gram-negative septicemia . Fifteen animals were randomized to control, Pseudomonas aeruginosa infusion at a rate of 2 x 10(7) colony forming units/20 kg/min (septic group), or Pseudomonas infusion combined with DFO pretreatment at a dose of 80 mg/kg/h (septic-treated group) . Three of six septic-treated animals became severely hypotensive and died during the course of the experiment as opposed to none of six septic animals . Surviving septic-treated animals were significantly hypotensive (60 +/- 24 mmHg mean arterial pressure) compared to septic (122 +/- 9 mmHg) and control (109 +/- 8 mmHg) animals . DFO did not improve respiratory function (e.g., pO2) or morphology in septic animals . We conclude that iron-chelation therapy with DFO at the above dosage results in a significant deterioration in cardiovascular function in septic swine . Lung damage was not prevented. Pediatr Res, 1994 Mar, 35(3), 299 - 302 Effect of pH and CO2 on in vitro susceptibility of Pseudomonas cepacia to beta-lactams; Corkill JE et al.; Inhibition of Pseudomonas cepacia (but not Pseudomonas aeruginosa) by beta-lactams was decreased in 5% CO2 in air compared with air alone . The effect of CO2 and pH (range, 6.0 to 8.0) on beta-lactam susceptibility, beta-lactamase expression, and outer membrane proteins was studied in isolates recovered from the sputum of children with cystic fibrosis . Incubation in 5% CO2 decreased the activity of piperacillin, piperacillin/tazobactam, and ceftazidime, although isolates were still clinically sensitive (minimum inhibitory concentrations < 16 mg/L) . Cefpirome activity was markedly decreased from a minimum inhibitory concentration of 2.0 to greater than 64 mg/L . On highly buffered 3-(N-morpholino)-propane sulfonic acid media, beta-lactam susceptibility was eliminated at pH greater 7.5 . A 2- to 13-fold increase in beta-lactamase activity was demonstrated after growth in 5% CO2 compared with basal aerobic levels for 13 of 15 clinical isolates . beta-Lactamase activity did not vary significantly with pH . Addition of imipenem to media (2.0 mg/L) resulted in hyperproduction of beta-lactamase (180-fold) . Isoelectric points varied with cultural conditions, and all beta-lactamases detected were inhibited by clavulanate and tazobactam . Significant hydrolysis of piperacillin and ceftazidime could not be demonstrated . A 36-kD porin was present at all pH tested . Thus, our strains of Pseudomonas cepacia were markedly affected by cultural conditions not normally used in standardized susceptibility tests . However, such conditions may be encountered in the pathologically altered infected lung in cystic fibrosis. FEMS Microbiol Lett, 1994 Mar 1, 116(3), 307 - 13 Elastase gene expression in non-elastase-producing Pseudomonas aeruginosa strains using novel shuttle vector systems; Ishii T et al.; In order to determine whether non-elastase-producing strains of Pseudomonas aeruginosa such as N-10, PA103 and IFO3080 can express foreign elastase genes, we introduced elastase genes from P . aeruginosa IFO3455 (elastase-producing) as well as from PA103 and N-10 into non-elastase-producing P . aeruginosa strains . Results suggested that gene expression, secretion, and precursor processing systems of elastase were essentially normal in P . aeruginosa N-10 and IFO3080 . Our studies using various elastase genes showed that both the elastase structural gene and 5'-upstream regions of P . aeruginosa PA103 were also normal . This was confirmed by the finding that P . aeruginosa N-10 and IFO3080 which carry the PA103 elastase gene produced elastase . Several deleted or chimeric genes were constructed using the 5'-upstream regions of elastase genes from P . aeruginosa N-10 or PA103 and studies of expression revealed that two individual DNA bases seem to be important in suppressing P . aeruginosa N-10 elastase gene expression . Possible reasons for the lack of elastase expression in these non-elastase-producing strains are discussed. Toxicology, 1994 Feb 28, 87(1-3), 69 - 83 Pore-forming Pseudomonas aeruginosa cytotoxin; Xiong G et al.; Pseudomonas aeruginosa procytotoxin protein is processed C-terminally during bacterial autolysis to generate the active 29-kDa cytotoxin molecule . Binding to target cell membranes is dependent upon Cys23 and Cys215 and a domain flanked to Cys215 . On rabbit erythrocytes, cytotoxin binds to a 28-kDa peptide of a glycoprotein, its N-terminus shows high homology to channel integral membrane protein CHIP28 . At concentrations of more than 3 x 10(-9) M, cytotoxin increases plasma membrane permeability of most eucaryotic cells investigated . The role of cytotoxin in the formation of pores with a diameter of 2 nm on mammalian cells is discussed . The cytotoxin effects are coordinated with other pseudomonal products and the resultant concept of pathogenesis is presented. J Biol Chem, 1994 Feb 18, 269(7), 4872 - 7 Identification of amino acid residues involved in the activity of phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase . A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa; May TB et al.; Phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase (PMI-GMP), which is encoded by the algA gene, catalyzes two noncontiguous steps in the alginate biosynthetic pathway of Pseudomonas aeruginosa; the isomerization of D-fructose 6-phosphate to D-mannose 6-phosphate and the synthesis of GDP-D-mannose and PPi from GTP and D-mannose 1-phosphate . Amino acids that are required for the GMP enzyme activity were identified through site-directed mutagenesis of the algA gene . Mutation of Lys-175 to arginine, glutamine, or glutamate produced an enzyme whose Km for D-mannose 1-phosphate was 470-3,200-fold greater than that measured for the wild type enzyme . In addition, these mutant enzymes had a lower Vmax for the GMP activity as compared with the wild type PMI-GMP . These results indicate that Lys-175 is primarily involved in the binding of the substrate D-mannose 1-phosphate, although it is likely that other residues are required for the specificity of binding . Mutation of Arg-19 to glutamine, histidine, or leucine resulted in a 2-fold lower Vmax for the GMP enzyme activity and a 4-7-fold increase in the Km for GTP as compared with the wild type enzyme . Thus, it appears that Arg-19 functions in the binding of GTP . In addition, chymotryptic digestion of PMI-GMP showed that the carboxyl terminus is critical for PMI activity but not for GMP activity . Taken together, these results support the hypothesis that the bifunctional PMI-GMP protein is composed of two independent enzymatic domains. Biochemistry, 1994 Feb 15, 33(6), 1545 - 54 Changes in the catalytic properties of p-hydroxybenzoate hydroxylase caused by the mutation Asn300Asp; Palfey BA et al.; By site-directed mutagenesis, we have changed Asn300 to Asp in p-hydroxybenzoate hydroxylase (PHBH; EC 1.14.13.2) from Pseudomonas aeruginosa . In the wild-type (WT) enzyme, residue 300 is in contact with the isoalloxazine ring of the active-site FAD; in the Asn300Asp mutant, this side chain has moved by about 5 A, altering the protein structure {Lah, M.S., Palfey, B.A., Schreuder, H.A., & Ludwig, M.L . (1994) Biochemistry (following paper in this issue)} . The structural changes are responsible for profound catalytic and dynamic effects . The flavin of PHBH is reduced by NADPH in the first half of catalysis . The mutation has decreased this rate 330-fold, apparently by affecting the reactive orientation of the isoalloxazine and pyridine rings . Furthermore, the redox potential of the flavin is lower in the mutant enzyme than in WT by 20-40 mV . The reduced flavin of PHBH reacts with O2 to form a flavin C(4a)-hydroperoxide, which is the species that transfers oxygen to the aromatic substrate . Previous studies indicated that the enzyme promotes the hydroxylation reaction in part by activating the substrate through lowering the phenolic pKa . The Asn300Asp mutant does not lower the substrate pKa . As a consequence of this, and also an enhanced stability of the flavin C(4a)-hydroperoxide, the hydroxylation is 50-fold slower in the mutant than in WT . However, despite the slow rate of the hydroxylation reaction, no H2O2 is formed by the competitive elimination reaction . The kinetic stability of the flavin C(4a)-hydroxide formed by the hydroxylation was also enhanced by the mutation . By studying the effects of the inhibitor azide on the oxidative sequence, we were able to conclude that the inhibitory site is readily accessible to solvent; azide binding at a second site slowly displaces the substrate from the reduced enzyme . The mutation has profoundly slowed the rates of ligand binding to the enzyme . Kinetic studies of binding indicated the presence of several enzyme conformations . Thus, the mutation of this one residue interferes with the orientation of pyridine nucleotide and flavin during reduction, stabilizes flavin C(4a) intermediates, prevents substrate ionization, and alters the rates and strengths of ligand binding. Biochemistry, 1994 Feb 15, 33(6), 1425 - 32 Unique environment of Trp48 in Pseudomonas aeruginosa azurin as probed by site-directed mutagenesis and dynamic fluorescence spectroscopy; Gilardi G et al.; Two mutants of the blue copper protein azurin from Pseudomonas aeruginosa, Ile7Ser and Phe110Ser, were prepared . The mutations were aimed at affecting the mobility and the fluorescence properties of Trp48, the only tryptophan residue present, which in the wild-type protein is located in a highly hydrophobic and rigid environment . EPR, UV-vis, and NMR spectroscopy show that the copper binding site and the overall structure of the wild-type protein are preserved and that structural effects occur only on a local scale . Steady-state fluorescence spectra of both mutants, particularly in the copper-free form, show that tryptophan fluorescence is dramatically affected by the introduction of a polar residue close to it . The emission maximum is red-shifted and dependent on the excitation wavelength . This indicates a loosening of the matrix around the indolyl side chain and an increase of the effective dielectric constant of the microenvironment . Time-resolved fluorescence spectroscopy also shows substantial changes in the fluorescence lifetimes and in the distribution of the lifetimes of the mutants; these variations are interpreted in terms of a change in solvation of the Trp48 side chain. Proc Natl Acad Sci U S A, 1994 Feb 15, 91(4), 1366 - 70 Pseudomonas aeruginosa possesses homologues of mammalian phenylalanine hydroxylase and 4 alpha-carbinolamine dehydratase/DCoH as part of a three-component gene cluster; Zhao G et al.; Pseudomonas aeruginosa possesses a multigene operon that includes phenylalanine hydroxylase (PhhA; phenylalanine 4-monooxygenase, EC 1.14.16.1) . phhA encodes PhhA (M(r) = 30,288), phhB (M(r) = 13,333) encodes a homologue of mammalian 4 alpha-carbinolamine dehydratase/homeodomain protein transregulator, and phhC encodes an aromatic aminotransferase (M(r) = 43,237) . The reading frames specifying phhB and phhC overlap by 2 bases . The P . aeruginosa PhhA appears to contain iron and is pterin dependent . Unlike the multimeric mammalian hydroxylase, the native P . aeruginosa enzyme is a monomer . The P . aeruginosa PhhA is homologous with mammalian PhhA, tryptophan hydroxylase, and tyrosine hydroxylase . Expression of PhhA from its native promoter required phhB . This may suggest a positive regulatory role for phhB, consistent with the dual catalytic and regulatory roles of the corresponding mammalian homologue. Gene, 1994 Feb 11, 139(1), 77 - 81 Construction and expression in Escherichia coli of hybrid genes composed of sequences encoding diphtheria toxin and human CD4 receptor; Zdanovsky AG et al.; Derivatives of natural toxins possessing substituted receptor-recognition domains of different specificities can be used as instruments for the selective elimination of target cells . We have constructed two different types of hybrid genes that encode proteins composed of diphtheria toxin (DT) lacking the C-terminal residues that mediate toxin binding fused with the N-terminal region of human CD4 (Lys10 to Glu152) . One of these hybrids encodes a protein with CD4 at the N terminus, while the other encodes a protein with CD4 at the C terminus . The stability of these two proteins was dramatically different . We could not detect a full-size product when the first construct was expressed in Escherichia coli . In contrast, proteins encoded by the second construct were more stable . In the latter case, the amount of full-size hybrid protein was 1-2% of the total cell protein . We speculate on the involvement of the region that resembles the processing site of Pseudomonas aeruginosa exotoxin A in the proteolytic degradation of the product encoded by the first type of hybrid. J Bacteriol, 1994 Feb, 176(3), 553 - 62 Genetic rearrangement associated with in vivo mucoid conversion of Pseudomonas aeruginosa PAO is due to insertion elements; Sokol PA et al.; The conversion of Pseudomonas aeruginosa PAO to the mucoid phenotype has been reported for a chronic pulmonary infection model in rats (D . E . Woods, P . A . Sokol, L . E . Bryan, D . G . Storey, S . J . Mattingly, H . J . Vogel, and H . Ceri, J . Infect . Dis . 163:143-149, 1991) . This conversion was associated with a genetic rearrangement upstream of the exotoxin A gene . To characterize the genetic rearrangement, the region upstream of the toxA gene was cloned from PAO, PAO-muc (a mucoi