The Pseudomonas aeruginosa initiation factor IF-2 is responsible for formylation-independent protein initiation in P. aeruginosa

Formylation of the initiator methionyl-tRNA (Met-tRNAfMet) was generally thought to be essential for initiation of protein synthesis in all eubacteria based on studies conducted primarily in Escherichia coli. However, this view of eubacterial protein initiation has changed because some bacteria have been demonstrated to have the capacity to initiate protein synthesis with the unformylated Met-tRNAfMet. Here we show that the Pseudomonas aeruginosa initiation factor IF-2 is required for formylation-independent protein initiation in P. aeruginosa, the first bacterium shown to have the ability to initiate protein synthesis with both the initiator formyl-methionyl-tRNA (fMet-tRNAfMet) and Met-tRNAfMet. The E. coli IF-2, which participates exclusively in formylation-dependent protein initiation in E. coli, was unable to facilitate utilization of Met-tRNAfMet in initiation in P. aeruginosa. However, the E. coli IF-2 was made to function in formylation-independent protein initiation in P. aeruginosa by decreasing the positive charge potential of the cleft that binds the amino end of the amino acid attached to the tRNA. Furthermore increasing the positive charge potential of this cleft in the P. aeruginosa IF-2 prevented the protein from participating in formylation-independent protein initiation. Thus, this is the first demonstration of a eubacterial IF-2 with an inherent capacity to facilitate utilization of Met-tRNAfMet in protein initiation, discounting the dogma that eubacterial IF-2 can only allow the use of fMet-tRNAfMet in protein initiation. Furthermore these findings give important clues to the basis for discriminating the initiator Met-tRNA by IF-2 and for the evolution of alternative mechanisms for discrimination.     

Pharmacodynamic and protective properties of a murine lipopolysaccharide-specific monoclonal antibody in experimental Pseudomonas aeruginosa pneumonia in mice.

We employed a Pseudomonas aeruginosa mouse pneumonia model to evaluate the ability of a murine monoclonal antibody (MAb) specific for the O-side chain of P. aeruginosa Fisher Immunotype-1 lipopolysaccharide (LPS) to achieve and sustain therapeutic levels in plasma and lung tissue, reduce bacterial populations in the lung, and prevent pneumonia-associated mortality. An IgG3 MAb (Y1-5A4) administered to mice i.v. over a dose range of 125-1,000 micrograms/mouse produced plasma and lung tissue levels at 2 hr of 61-507 micrograms/ml and 4.3-150 micrograms/g, respectively. The 1,000 micrograms MAb dose reduced bacterial counts in lung tissue (log10 cfu/g +/- S.D.) and blood (log10 cfu/ml +/- S.D.) 20 hr post-treatment (18 hr post-challenge) from 10.00 +/- 0.66 to 7.66 +/- 0.91 (P less than 0.01) and from 4.39 +/- 0.81 to less than 3.0, respectively. Administration of MAb to mice in doses of 125-500 micrograms 2 hr prior to a 3 x 50% lethal bacterial challenge produced significant protection against death, with a calculated 50% protective dose of 167 micrograms. Protection was noted following administration of 1,000 micrograms of MAb up to 6 hr after bacterial challenge (P less than 0.05, compared with untreated control). Histological examination of lung tissue from infected mice revealed less acute inflammation, necrosis, and hemorrhage in MAb-treated compared with untreated control animals and greater localization of Pseudomonas antigen within the phagocytic cells in alveolar space. These findings document the in vivo therapeutic efficacy of an LPS-specific IgG MAb in a murine model of acute P. aeruginosa pneumonia, based in part upon the achievability of effective MAb concentrations in plasma and lung tissue.     

短期菲胁迫对大豆幼苗超氧化物歧化酶活性及丙二醛含量的影响

研究了不同浓度(0-200μg.g^-1)菲胁迫和恢复培养后大豆幼苗生长、超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量的变化．结果表明,200μg.g^-1菲处理5d后大豆幼苗生长受到抑制,但幼苗恢复培养后经短暂停滞期后仍可恢复生长．菲污染对大豆幼苗SOD活性变化的剂量—效应关系的作用形式比较复杂,胁迫2d时为线性关系,胁迫5d和8d时为抛物线型．在菲处理前期(2d),幼苗SOD活性被100和200μg.g^-1菲显著诱导[分别为对照的1．15倍(P＜0．05)和1．26倍(P＜0．01)]．菲暴露8d时,SOD活性显著降低,200μg.g^-1菲处理组SOD活性为对照的88％(P＜0．05)．菲处理5d后恢复培养2d和4d,50和100μg.g^-1菲处理组幼苗SOD活性得到恢复,而200μg.g^-1菲处理组幼苗SOD活性仍明显高于对照(P＜0．05)．试验亦反映出,100和200μg.g^-1菲处理5d和8d,幼苗MDA含量均比对照显著增加(P＜0．05和P＜0．01)．可以认为,SOD活性可作为大豆幼苗遭受短期菲胁迫的生物标记物．     

Function of initiation factor 1 in the binding and release of initiation factor 2 from ribosomal initiation complexes in Escherichia coli.

1. Studies on the function of initiation factor 1 (IF-1) in the formation of 30 S initiation complexes have been carried out. IF-1 appears to prevent the dissociation of initiation factor 2 (IF-2) from the 30 S initiation complex. The factor has no effect on either the initial binding of IF-2 nor does it increase the amount of IF-2 dependent fMet-tRNA and GTP bound to the 30 S subunit. Bound fMet-tRNA remains stable to sucrose gradient centrifugation even in the absence of IF-1. 2. It is postulated that the presence of IF-2 on the 30 S complex is necessary so that at the time of junction with the 50 S subunit to form a 70 S complex, the 70 S-dependent GTPase activity of IF-2 can hydrolyze GTP. This hydrolysis provides a means by which GTP can be removed to facilitate formation of a 70 S initiation complex active in peptidyl transfer. In support of this postulate, it was observed that 30 S initiation complexes formed in the absence of IF-1 could be depleted of their complexes were still able to accept 50 S subunits to form 70 S complexes which could still donate fMet-tRNA into peptide linkages. These results indicate that 30 S complexes lacking GTP do not require IF-2 for formation of active 70 S complexes. 3. IF-1, which is required to prevent dissociation of IF-2 from the 30 S initiation complex, is also required for release of IF-2 from ribosomes following 70 S initiation complex formation. The mechanisms of the release of IF-2 has been studied in greater detail. Evidence is presented which rules out the presence of a stable IF-2 GDP complex on the surface of the 70 S ribosome following GTP hydrolysis and of any exchange reactions between IF-1 and guanine nucleotides necessary for effecting the release of IF-2. IF-2 remains on the 70 S initiation complexes after release of guanine nucleotides and can be liberated solely by addition of IF-1.     

Effects of PGE1 or PGE2 on luteal function in pseudopregnant rats

Effects of PGE1 or PGE2 on luteal function were studied in 163 pseudopregnant rats. PGE1 (10, 100, or 300 micrograms) given intrauterine every 6 hr did not shorten pseudopregnancy (P greater than 0.05), however, the same doses of PGE2 given intrauterine every 6 hr advanced luteolysis (P less than 0.05). PGE1 (100 or 300 micrograms) given every 4 hr intramuscular maintained levels of progesterone in peripheral blood above controls (P less than 0.05) while 100 or 300 micrograms of PGE2 hastened the decline in progesterone (P less than 0.05). The antiluteolytic effect of PGE1 was not via an inhibition of PGF secretion (P greater than 0.05) by the uterus or by induction of ovulation in treated animals. Moreover, PGE1 (100, 200, or 500 micrograms) given intramuscular every 4 hr from day 4 of pseudopregnancy until the next proestrus delayed luteal regression around 3 days (P less than 0.05). PGE2 at doses of 100, 200, or 500 micrograms every 4 hr given intramuscular consistently shortened pseudopregnancy (P less than 0.05). Lower doses were without effect (P greater than 0.05). Based on the above data it is concluded that PGE2 is consistently luteolytic whereas PGE1 is not luteolytic in pseudopregnant rats and that PGE1 may be an antiluteolysin.     

Purification and characterization of six cytochromes P-450 from hepatic microsomes of immature female rats

Six rat hepatic cytochromes P-450, named P-450IF-1-6, were purified from hepatic microsomes of immature female rats by high-performance liquid chromatography with anion-exchange, cation-exchange, and hydroxylapatite columns. The purified forms, except for P-450IF-4, gave a single protein-staining band on SDS-polyacrylamide gel electrophoresis, with a minimum molecular weight of 50,000 for P-450IF-1, 49,000 for P-450IF-2, 47,000 for P-450IF-3, 53,500 for P-450IF-5, and 54,000 for P-450IF-6. The CO-reduced spectral maximum of these forms was at 450 nm for P-450IF-1, 448 nm for P-450IF-2, 451 nm for P-450IF-3, 449 nm for P-450IF-4, 449 nm for P-450IF-5, and 450 nm for P-450IF-6. All of these cytochromes had the low-spin state of heme in the oxidized form. P-450IF-4 had high metabolic activity for both benzphetamine and 7-ethoxycoumarin. P-450IF-5 had moderate activity toward 7-ethoxycoumarin. P-450IF-3 catalyzed the hydroxylation of testosterone at the 7 alpha-position effectively, but the other forms did not hydroxylate testosterone. Analysis of the NH2-terminal sequence showed that P-450IF-1, 2, 3, 5, and 6 differed structurally from each other. The sequences of P-450IF-1 and IF-2 were somewhat homologous, but the NH2-terminal sequences of the other forms were all different. Based on these results, we concluded that P-450IF-1 corresponded to one of the phenobarbital-inducible forms in male rat liver. P-450IF-2 was a female-specific form and its concentration was low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppressor T cells regulating the cell-mediated immune response to Pseudomonas aeruginosa can be generated by immunization with anti-bacterial T cells

We previously demonstrated that immunization with low (10 micrograms) doses of high m.w. polysaccharide from the gram-negative bacterium Pseudomonas aeruginosa generates T cells that suppress the ability of antibacterial T cells (Tab) to protect against bacterial infection. The current studies indicate that Ts cells with properties identical to those elicited by low dose polysaccharide immunization can be generated by immunization with Tab. Tab-elicited Ts cells can abrogate in vivo induction and in vitro and in vivo expression of antibacterial T cell activity. Tab-elicited Ts are Ag-specific and H-2 restricted in their suppressor activity. Non-immune T cells fail to elicit suppressor activity. These studies provide additional evidence that the protective T cell response to P. aeruginosa is controlled by a network of T cells that are probably recognizing idiotypic determinants on P. aeruginosa-immune T and B cells.     

Protection against gram-negative bacteremia in neutropenic mice with recombinant granulocyte-macrophage colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates production of neutrophils in bone marrow and may decrease the incidence of infection during neutropenia. We evaluated the protective role of recombinant GM-CSF against Pseudomonas aeruginosa challenge in neutropenic mice. CD-1 mice treated with cyclophosphamide on days 1 and 2 of the experiment were given GM-CSF (1, 2, or 4 micrograms/day) starting at day 4 of the experiment according to the following protocol: 1) 1 microgram of GM-CSF 2 hr and 24 hr after challenge; 2) 1 microgram 24 hr before challenge, 2 hr and 24 hr after challenge; 3) 2 micrograms injected 24 hr before and 2 hr after challenge; 4) 2 micrograms given 24 hr before and 2 micrograms given 2 hr and 24 hr after challenge; 5) 4 micrograms administered 2 hr and 24 hr after challenge; and 6) saline and bovine albumin controls. The number of blood neutrophils by days 4 and 5 was similar for GM-CSF-treated and untreated animals. Survival was significantly greater in animals given 2 micrograms of GM-CSF at 24 hr before and at 2 hr and 24 hr after challenge with Pseudomonas. Neutrophils and splenic macrophages obtained from GM-CSF-treated mice (2 micrograms/animal) produced significantly greater amounts of O2- (204 +/- 36 nmoles/10(5) cells) than controls (21 +/- 10 nmoles/10(5) cells). Additionally, neutrophils and macrophages from GM-CSF-treated mice killed significantly more bacteria (P. aeruginosa) in vitro and had a greater number of C3b and Fc receptors (78 +/- 12% and 89 +/- 8%) than did cells obtained from control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human monoclonal antibodies to Pseudomonas aeruginosa produced by EBV-transformed cells

Numerous lymphoblastoid cell lines (LCLs) which secreted antibodies against Pseudomonas aeruginosa (all Fisher's immunotypes and Homma's immunotype 1) were established by Epstein-Barr virus (EBV)-transformation of lymphocytes. Five LCLs were established as long-term culture lines and their properties were determined. These LCLs produced monoclonal antibodies to Fisher's immunotype 1 and 4 and Homma's immunotype 1, and their immunoglobulin classes were IgM, IgG, and IgA. We found that three monoclonal antibodies (G3-1, H7-2, and E10-1) among them successfully protected mice from the corresponding immunotype of P. aeruginosa infection. Their protective dose (PD50) values were 0.5, 2.6, and 3.1 micrograms immunoglobulin/mouse. These human monoclonal antibodies against P. aeruginosa prepared by EBV-transformation method will be a valuable aid for the treatment for severe P. aeruginosa infections.     

Selective medium for Pseudomonas aeruginosa that uses 1,10-phenanthroline as the selective agent.

The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa.     

Selective medium for Pseudomonas aeruginosa that uses 1,10-phenanthroline as the selective agent

The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa.     

Structure-function relationship in Escherichia coli initiation factors. Biochemical and biophysical characterization of the interaction between IF-2 and guanosine nucleotides

Equilibrium dialysis and protection from heat inactivation and proteolysis show that initiation factor 2 (IF-2) interacts not only with GTP but also with GDP and that its conformation is changed upon binding of either nucleotide. The apparent Ka (at 25 degrees C) for the IF-2 X GDP and IF-2 X GTP complexes was 8.0 X 10(4) and 7.0 X 10(3) M(-1), respectively. The lower affinity for GTP is associated with a more negative delta S0. The interaction, monitored by 1HNMR spectroscopy, is characterized by fast exchange and results in line broadening and downfield shift of the purine C-8 and ribose C-1' protons of GTP as well as of the beta, gamma-methylene protons of (beta-gamma-methylene)guanosine 5'-triphosphate. The interaction of guanosine nucleotides with IF-2 requires an H bond donor (or acceptor) group at position C-2 of the purine and involves the beta- and/or gamma-phosphate of the nucleotide while the ribose 2'-OH group or the integrity of the furan ring are less critical. IF-2 binds to ribosomal particles with decreasing affinity: 30 S greater than 70 S greater than 50 S. GTP and GDP have no effect on the binding to 70 S. GTP stimulates the binding to the 30 S and depresses somewhat the binding to the 50 S subunits; GDP has the opposite effect. These results seem to rule out that the release of IF 2 from 70 S is due to a "GDP-conformation" of the factor incompatible with its permanence on the ribosome. The rate and the extent of 30 S initiation complex formation are approximately 2-fold higher with IF-2 X GTP than with IF-2 alone. At low concentrations of IF-2 and 30 S subunits, GDP inhibits this reaction, acting as a strong competitive inhibitor of GTP (Ki = 1.25 X 10(-5)m) and preventing IF-2 from binding to the ribosomal subunit.     

Effect of Escherichia coli initiation factors on the kinetics of N-Acphe-tRNAPhe binding to 30S ribosomal subunits. A fluorescence stopped-flow study

The mechanism of binding of N-AcPhe-tRNAPhe (yeast) to poly(U)-programmed Escherichia coli 30S ribosomal subunits and the effect of individual initiation factors (IF-1, IF-2, and IF-3) and GTP on this process have been studied by fluorescence stopped-flow kinetic measurements. The formation of the ternary complex was followed by an increase of both intensity and polarization of the fluorescence of a proflavin label located in the anticodon loop of the tRNA. The effect of the initiation factors and GTP is to increase the velocity of ternary complex formation (about 400-fold at 7 mM Mg2+). In the presence of the three initiation factors and GTP the formation of the ternary complex could be resolved into two partial reactions: a fast apparently second-order step (k12 = 5 x 10(6) M-1 s-1, k21 = 1.4 s-1) followed by a slow rearrangement step (k23 less than or equal to 0.1 s-1). The data suggest a mechanism in which the ternary complex is formed by at least two rearrangements of an initially formed preternary complex. The accelerating effects of both IF-2 and IF-3 can be understood by assuming a synergistic allosteric action of the factors on the 30S ribosomal subunit, whereas IF-1 appears to act indirectly by influencing the other two factors.     

C-390 as sole selective agent for isolation of Pseudomonas aeruginosa from hospital waste water

Pseudomonas aeruginosa was recovered (in numbers ranging from 10(2) to 10(5) colony-forming units per millilitre) from heavily contaminated hospital waste water when grown at 41.5 degrees C on a differential medium agar containing 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan (C-390) at a final concentration of 30 micrograms/mL. The medium appeared to be highly selective for P. aeruginosa with 95-100% of all colonies isolated from four different hospital waste waters being identified as P. aeruginosa. Many strains of P. aeruginosa isolated from hospital waste waters failed to hydrolyse casein when grown on skim milk agar and this medium appeared to restrict pigment production to only pyoverdin (detectable only under ultraviolet light). However, most strains were capable of casein hydrolysis when grown on a modified skim milk medium.     

T cells recognizing polysaccharide-specific B cells function as contrasuppressor cells in the generation of T cell immunity to Pseudomonas aeruginosa

The cellular interactions involved in the development of T cell-mediated immunity to Pseudomonas aeruginosa have been examined. T cell immunity can be generated by immunizing mice with 10 micrograms of P. aeruginosa polysaccharide (PS) plus the antimitotic agent vinblastine sulfate. Vinblastine is required to inactivate a population of Ts cells generated by immunization with 10 micrograms of PS alone. Immunization with either live bacteria or with higher dose (50 micrograms) of PS without vinblastine also generates T cell immunity; these protocols activate a population of Lyt-1+, 2-, I-J+ T cells which, like vinblastine, counteract the effect of Ts cells. Immunization with 10 micrograms PS alone fails to activate this T cell subpopulation. When administered at the time of immunization, this subpopulation can render the tolerogenic 10-micrograms immunization protocols immunogenic. Like previously described contrasuppressor T cells, this T cell subpopulation exhibits an affinity for the lectin Vicia villosa. We have determined, however, that the T cells that act as contrasuppressor cells in this system are directly activated by PS-immune B cells and not by PS Ag. Furthermore, their activity can be removed by adsorption to PS-specific B cell hybridomas. Our studies indicate an important role for B cells in the development of T cell immunity to P. aeruginosa and suggest that a complex idiotype network controls the development of this response.     

The binding of dansylated initiation factor 3 to the 30-S subunit of Escherichia coli: a fluorimetric and biochemical study

Initiation factor 3 (IF-3) has been labelled using dansyl (1-dimethylaminonaphthalene-5-sulphonyl) chloride under conditions designed to preserve the biological activity of the factor. The sites of modification of the IF-3 have been determined by peptide mapping and sequencing: about six lysines (73, 80, 91, 96, 99, 112) react in various proportions. However, if an IF-3 molecule bears more than one dansyl group on average then its activity is lost. The extent of incorporation is proportional to the amount of dansyl chloride used in the reaction. Spectrofluorimetric analysis of the dansyl-IF-3 leads to the following conclusions. (a) The motion of the dansyl label does not change greatly upon binding to the 30-S subunit. (b) The label is not close enough to any tryptophan group of the ribosome in the 30-S-subunit . IF-3 complex to allow energy transfer. (c) The IF-3 chain is folded so as to bring the tyrosine groups close to the dansyl-binding sites. (d) The stoichiometry of the binding of IF-3 to 30-S ribosomal subunits is close to 1:1 and the binding constant is 2 x 10(7) M-1. IF-3 also binds non-covalently the fluorescent indicator 8-anilinonaphthalene 1-sulphonate (ANS) with an apparent binding constant of approximately 8000 M-1. An interaction between ANS and poly(A-U-G), both bound to IF-3, was demonstrated. Combining these results with those for dansyl-IF-3 leads to a model for the interaction between IF-3 and the 30-S subunit involving a combination of 'hydrophobic' and electrostatic attraction between the factor and ribosomal RNA.     

Establishment of stable cell lines producing anti-Pseudomonas aeruginosa monoclonal antibodies and their protective effects for the infection in mice.

Human-human hybridomas producing monoclonal antibodies (MoAbs) specific for five major serotypes of Pseudomonas aeruginosa were developed by fusing P. aeruginosa primed and Epstein-Barr virus-transformed cells with human myeloma P109 cells using polyethyleneglycol. The MoAbs which were produced by the hybridomas were protective against lethal intraperitoneal (i.p.) challenge of P. aeruginosa (10 LD50) in mice. The 50% effective dose (ED50) values of MoAbs ranged from 0.5 to 10.2 micrograms/mouse and were 26 to 240 times more protective than a commercial human IgG preparation. MoAb administration to mice promoted bacterial clearance in peritoneal cavity, and prevented bacterial invasion into blood in the way of increasing both the number of bacteria trapped by a macrophage and the ratio of macrophages that trapped bacteria. MoAbs also showed protective effects against lethal infection of P. aeruginosa in the mice which were decreased in polymorphonuclear cells (PMN) by cyclophosphamide (CY). All MoAbs showed serotype-specific binding to the clinical isolates of P. aeruginosa as well as to the immunized strains. The hybridoma cell lines maintained their capacity to produce MoAb continuously for more than 12 months and produced 10 to 60 micrograms MoAbs per 10(6) cells in 24 hr. It is practicable to use these cell lines for large-scale production of anti-P. aeruginosa MoAbs and such MoAbs must be useful for the therapeutics of patients with P. aeruginosa infection.