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1.
Sequentially collected sera from cats chronically immunized with Pseudomonas aeruginosa lipopolysaccharide (LPS) serotype 5 were assessed for their effect on phagocytosis by alveolar macrophages from nonimmunized cats. Phagocytosis was measured by incubating macrophage monolayers for 20 min in the presence of 3H-labeled bacteria and 5% serum from control or immunized animals. Sustained phagocytic inhibitory activity developed in the sera of eight of 11 immunized cats (mean inhibition ranged from 30% to 73%) after 13 weekly immunizations. The activity was specific because phagocytosis of Staphylococcus aureus and P. aeruginosa of another serotype (type 6) was unimpaired. An enzyme-linked immunosorbent assay showed an increase in the amount of LPS-specific IgG in postimmunization sera from the eight cats with inhibitory activity. The IgG appeared to be serotype specific because significantly higher titers were obtained against LPS serotype 5 than LPS serotype 6. The results suggest that phagocytic inhibitory activity in sera from LPS-immunized cats may be due to anti-LPS IgG. 相似文献
2.
The nature of the humoral immune mechanisms involved in the protection induced after local immunization with a temperature-sensitive (ts) mutant of Pseudomonas aeruginosa was investigated. We had previously shown that intranasal (i.n.) immunization of granulocytopenic mice protected the animals from lethal pulmonary challenge with P. aeruginosa, whereas mice immunized intraperitoneally were unprotected. Intranasal immunization induced high levels of anti- P. aeruginosa IgG and IgA in the lower respiratory tract, whereas only modest levels of IgG (and no IgA) could be detected in lung lavage fluids from mice immunized by the intraperitoneal (i.p.) route with ts mutant E/9/9. Plasma anti- P. aeruginosa IgG levels after i.n. immunization were lower than those observed after i.p. immunization with similar doses of the ts mutant. The main contribution to the protection induced when mice are immunized intranasally appears to be from IgA in the pulmonary secretions, although other immune mechanisms cannot be discounted. 相似文献
3.
Pseudomonas aeruginosa outer membrane protein F was purified by extraction from polyacrylamide gels of cell envelope proteins of an Escherichia coli strain expressing the cloned gene for protein F. Antisera directed against protein F purified from P. aeruginosa PAO1 reacted with this E. coli strain by immunofluorescence assay and immunoblotting, whereas these antisera were nonreactive with E. coli strains lacking the Pseudomonas protein F gene. The protein F purified from this E. coli strain was used to immunize mice by intramuscular injection of 10 µg of protein F preparation on days 1 and 14, followed by burn and challenge of the mice on day 28. As compared with control mice immunized with E. coli K-12 lipopolysaccharide, immunization with the E. coli-derived protein F afforded significant protection against subsequent challenge with heterologous Fisher-Devlin immunotype 5 and 6 strains of P. aeruginosa. Antisera from mice immunized with the E. coli-derived protein F reacted at bands corresponding to protein F and 2-mercaptoethanol-modified protein F upon immunoblotting against cell envelope proteins of the PAO1, immunotype 5, and immunotype 6 strains of P. aeruginosa and the E. coli strain containing the cloned F gene, but failed to react at these sites in an E. coli strain lacking the F gene. These data demonstrate that P. aeruginosa protein F produced in E. coli through genetic engineering techniques retains its vaccine efficacy in the complete absence of any P. aeruginosa lipopolysaccharide. 相似文献
4.
Recombinant outer membrane protein F of Pseudomonas aeruginosa was purified by extraction from polyacrylamide gels of cell envelope proteins of an Escherichia coli strain expressing the cloned gene for protein F. Rats were immunized intramuscularly with 25 g of recombinant protein F adsorbed to aluminum hydroxide adjuvant on days 1, 14, and 28 and then challenged on day 42 via intratracheal inoculation of agar beads containing cells of a clinical isolate of P. aeruginosa. On day 49 the lungs were examined macroscopically for the presence and severity of lesions and submitted for quantitation of the bacteria present. The recombinant protein F vaccine afforded significant protection against subsequent challenge with P. aeruginosa in the immunized rats, as compared with control rats immunized with bovine serum albumin. Antisera from the recombinant protein F-immunized rats mediated opsonophagocytic uptake by human polymorphonuclear leukocytes of wild-type cells of P. aeruginosa but exhibited no opsonic activity against a protein F-deficient mutant of P. aeruginosa. The antisera to recombinant protein F did not promote complement-mediated bacteriolysis of P. aeruginosa. These data demonstrate that recombinant P. aeruginosa protein F has efficacy as a protective vaccine in a rat model of chronic pulmonary infection. 相似文献
5.
A comparative study has been carried out with FDP aldolases from Escherichia coli 518 and Lactobacillus casei ATCC 7469, which had been purified 17.6- and 65-fold, respectively. The aldolase of L.casei was stable only in the presence of mercaptoethanol, whereas that of E.coli was strongly inhibited at low (1.0×10 –4
m) and activated at high concentrations (2.0×10 –1
m) of the same compound. p-Chloromercuric benzoic acid inhibited both aldolases, with 40% inhibition at 2×10 –5
m with E.coli aldolase against at 2×10 –4
m with L.casei aldolase. Significant differences were also observed in pH optima and Km values. E.coli aldolase exhibited a maximal activity at pH 9.0 and gave a Km value of 1.76×10 –3
m FDP with strong substrate inhibition above 7×10 –3
m, against pH 6.8–7.0 and a Km of 7.04×10 –3
m FDP for L.casei aldolase. Strong resistance of L.casei aldolase against inhibition by EDTA, Ca 2+ and Mn 2+ was observed compared with complete inhibition at concentrations of 20 mm, 40 mm and 20 mm, respectively, with E. coli aldolase. Polyacrylamide gel electrophoresis did not reveal any differences between the two enzyme preparations.The differences of the properties of FDP aldolases from different bacterial genera are discussed in relation to other Class II aldolases. 相似文献
6.
Summary The in vitro activity of a combination of colimycin and tetracycline against Pseudomonas aeruginosa and Escherichia coli was studied with the aid of tube dilution tests, Elek and Hilson's modification of the replica technique, and growth inhibition curves. The interaction between the two antibiotics was as a
rule of an additive nature, but a slight synergism was observed in some instances. The values of the minimum inhibitory concentrations
did not seem to influence the nature of the interaction. A considerable synergism observed in all tube dilution tests with E. coli strains is attributed partly to addition and partly to suppression of the growth of more colimycin-resistant bacteria by
the tetracycline.
Possible in vivo applications of this antibiotic combination are briefly discussed. 相似文献
7.
Incubation experiments using washed cells and toluene treated cells of Streptomyces garyphalus showed that O-acetyl- L-serine and hydroxyurea are intermediates in the biosynthesis of D-cycloserine. The formation of [ 14C]O-ureidoserine from O-acetyl- L-serine and hydroxyurea was demonstrated by incubating an enzyme solution with 14C-labelled substrates. Desalted cell-free extract catalyzed the conversion of O-ureido- D-serine to D-cycloserine in a reaction requiring ATP and Mg 2+. The results suggested the following pathway for D-cycloserine biosynthesis. 相似文献
8.
Moxalactam (LY127935), a novel beta-lactam antibiotic, was compared with semisynthetic penicillins, cephalosporins, and aminoglycosides
by the agar dilution method against 5,317 recent clinical isolates of facultative and anaerobic bactria. At 0.5 μg/ml, moxalactam
inhibited 90% of all Gram-negative bacilli tested except for Pseudomonas aeruginosa (81% inhibited by 32 μg/ml) and Acinetobacter calcoaceticus (88% inhibited by 32 μg/ml). More than 90% of Bacteroides fragilis and Staphylococcus aureus were inhibited by 4 μg/ml and 8 μg/ml, respectively. Moxalactam was at least 16-fold more active by weight than cephalothin,
cefamandole, and cefoxitin for Escherichia coli, Klebsiella pneumoniae, and Enterobacter species, and 2- to 4-fold more active than cefoxitin for B. fragilis. Moxalactam was 4-fold less active than cefamandole and cephalothin for S. aureus and 2- to 4-fold less active than piperacillin for P. aeruginosa. Moxalactam was as active or more active than the aminoglycosides for all facultative Gram-negative bacilli except for P. aeruginosa. Moxalactam was inhibitory (minimal inhibitory concentration <16 μg/ml) for 20/27 gentamicin-resistant isolates and 8/13
amikacin-resistant organisms. Moxalactam’s in vitro activity against Gram-negative bacilli is markedly superior to presently
available cephalosporins and, except for P. aeruginosa, is comparable to the aminoglycosides. 相似文献
9.
Astrocytes possess a concentrative l-ascorbate (vitamin C) uptake mechanism involving a Na +-dependent l-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellular l-ascorbate on the activity of this transport system. Initial rates of l-ascorbate uptake were measured by incubating primary cultures of rat astrocytes with l-[ 14C]ascorbate for 1 min at 37°C. We observed that the apparent maximal rate of uptake ( V
max) increased rapidly (<1 h) when cultured cells were deprived of l-ascorbate. In contrast, there was no change in the apparent affinity of the transport system for l-[ 14C]ascorbate. The increase in V
max was reversed by addition of l-ascorbate, but not D-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures with l-ascorbate did not affect uptake of 2-deoxy- D-[ 3H(G)]glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels. 相似文献
10.
Most Pseudomonas aeruginosa PAO mutants which were unable to utilize l-arginine as the sole carbon and nitrogen source ( aru mutants) under aerobic conditions were also affected in l-ornithine utilization. These aru mutants were impaired in one or several enzymes involved in the conversion of N 2-succinylornithine to glutamate and succinate, indicating that the latter steps of the arginine succinyltransferase pathway can be used for ornithine catabolism. Addition of aminooxyacetate, an inhibitor of the N 2-succinylornithine 5-aminotransferase, to resting cells of P. aeruginosa in ornithine medium led to the accumulation of N 2-succinylornithine. In crude extracts of P. aeruginosa an ornithine succinyltransferase ( l-ornithine:succinyl-CoA N 2-succinyltransferase) activity could be detected. An aru mutant having reduced arginine succinyltransferase activity also had correspondingly low levels of ornithine succinyltransferase. Thus, in P. aeruginosa, these two activities might be due to the same enzyme, which initiates aerobic arginine and ornithine catabolism.Abbreviations OAT
ornithine 5-aminotransferase
- SOAT
N 2-succinylornithine 5-aminotransferase
- Oru
ornithine utilization
- Aru
arginine utilization 相似文献
11.
A total of 416 samples comprising faecal samples from diarrhoeic cases of man, calves, sheep and goats, and urine samples from patients with urinary tract infections, were examined for the presence of enterobacteria of emerging pathogenic significance. Citrobacter freundii from 20, C. intermedius biotype-a from four, Serratia marcescens (serotype 05:H13, bactericin type 16) from one and Erwinia herbicola from two human stool samples were isolated. Only two urine samples yielded C. freundii. Citrobacter freundii was isolated from 10 and C. intermedius biotype-a from two calves. From sheep and goats, two isolates of C. freundii and three of C. intermedius biotype-a were obtained. None of these samples yielded Edwardsiella tarda or Yersinia enterocolitica. The examination of 99 toads and 145 wall lizards revealed that toads were reservoirs for C. freundii, C. intermedius biotype-a and Salmonella brijbhumi, whereas wall lizards were reservoirs for C. freundii, C. intermedius biotype-a, E. herbicola, Enterobacter cloacae and Salmonella spp. These bacteria were present in the range of 2.0 × 10 6 to 6.0 × 10 11 organisms per gram of intestinal contents. In addition, toads were carriers for Edwardsiella tarda (new serotypes 04167:H1 and 05159: nonmotile). None of the toads and wall lizards proved positive for C. intermedius biotype-b ( C. koseri), S. marcescens and Y. enterocolitica. C. freundii, C. intermedius biotype-a, E. herbicola and S. marcescens were resistant to penicillin and erythromycin whereas E. tarda isolates were also resistant to gentamycin, neomycin, colistin and sulfamethaxazole. 相似文献
12.
Enterobacteriaceae members are largely distributed in the environment and responsible for a wide range of bacterial infections in hospitalized patients. Pseudomonas aeruginosa (P. aeruginosa) causes severe nosocomial infections associated with severe inflammation due to its potent virulent factors including lipopolysaccharide (LPS). The aim of this study is to assess the bacterial LPS effect on Enterobacteriaceae biofilm and other virulence factors in vitro. The effect of P. aeruginosa LPS on biofilm formation of two other species of Enterobacteriaceae (Escherichia coli and Klebsiella pneumoniae) was assessed using a standard biofilm assay. PCR was performed on genes of biofilm and virulence factors. Expression of biofilm, type-1-fimbriae and serum resistance genes in treated and untreated cells was measured with RT-PCR. P. aeruginosa LPS has the ability to stimulate biofilm formation and stabilize the already formed biofilm significantly in all tested strains. In addition, LPS significantly increased the level of expression of Bss, FimH, and Iss genes when measured by RT-PCR. P. aeruginosa LPS has a direct stimulatory effect on the biofilm formation, type-1-fimbriae, and serum resistance in both E. coli and K. pneumoniae. So, the presence of P. aeruginosa in mixed infection with Enterobactereacea leads to increase their virulence. 相似文献
13.
Lipopolysaccharide (LPS) is the major surface antigen of Pseudomonas aeruginosa and elicits protective antibodies in animals. No cross reaction was observed between LPSs of P. aeruginosa International Antigenic Typing Scheme (IATS) 6 and 11 strains using rabbit polyclonal antibodies raised against the whole
cells. The O-polysaccharides (O-PSs) from IATS 6 and 11, the antigenic determinant of LPS, were directly coupled to bovine
serum albumin (BSA) by carbodiimide mediated condensation reaction. The molar ratios of saccharide repeating units to BSA
in the prepared conjugates were 15:1 and 26:1 for IATS 6 and 11 conjugates, respectively. Mice were immunized with the conjugates
emulsified with monophosphoryl lipid A (MPL), Freund, and Alum adjuvants. The conjugates emulsified with MPL adjuvant elicited
the highest IgM antibody, followed by Freund. While both MPL and Freund adjuvants elicited high IgG antibody. Good correlation
was observed between the IgG and IgM levels with the bactericidal activities of the sera against homologous strains. In addition,
immunization of mice with the prepared conjugates emulsified with MPL and Freund adjuvants provided high protection against
ten times the LD 50 of P. aeruginsoa IATS 6 and 11, which showed a good correlations with the IgG titer. 相似文献
14.
The genus Pimpinella contains rare phenylpropanoids. The 1-(E)-propenyl-2-hydroxy-5-methoxy benzene skeleton of these compounds is called pseudoisoeugenol. To study the biosynthesis of these compounds, we set up a tissue culture of Pimpinella anisum (PAD) that selectively promoted the production of epoxy-pseudoisoeugenol-(2-methylbutyrate), termed EPB. This compound served as the final molecule of the biosynthetic pathway in all labelling experiments conducted.The putative precursors were labelled with 13C or 14C. The incorporation of the label was followed by 13C-NMR-spectroscopy and liquid scintillation, respectively. Based on our labelling experiments as well as on enzymic reactions in a cell homogenate we proposed a genaral biosynthetic pathway for EPB. The biosynthetic sequence found was l-phenylalanine, trans-cinnamic acid, p-coumaric acid, p-coumaric aldehyde, p-coumaric alcohol, anol and trans-anethol. The biosynthetic step leading from trans-anethol to pseudo-isoeugenol involves migration of the side chain during the introduction of the second OH-group in the molecule (NIH-shift). The final biosynthetic steps to form EPB must be acylation and epoxidation of the propenyl double bond of pseudoisoeugenol.Abbreviations NMR
Nuclear Magnetic Resonance
- NOE
Nuclear Overhauser Effect 相似文献
15.
Ribosomal ribonucleic acid (RNA) and lipopolysaccharide (LPS) from P. aeruginosa were compared with respect to their protective activities in mice against an infection with P. aeruginosa. This study is concentrated on the protective activity of RNA. RNA isolated from purified ribosomes did not contain LPS as determined with the Limulus test. Injection of RNA with the adjuvant dimethyldioctadecylammonium bromide (DDA) protected mice against P. aeruginosa without inducing LPS-specific antibodies. C 3H/HeJ mice which are relatively insensitive to the protective activity of LPS could be protected with RNA. The protective activities of RNA and LPS from a mutant strain of P. aeruginosa, PAC 605, containing defective lipopolysaccharide, were compared with the protective activities of RNA and LPS from the parent strain, PAC IR. The protective activity of LPS from PAC 605 was 1000 fold lower than the protective activity of LPS from PAC IR. RNA preparations of both strains induced similar percentages of survival. The protective activity of ribosomal RNA from P. aeruginosa was nonspecific since mice were also protected against a heterologous serotype of P. aeruginosa and against Escherichia coli. RNA from ribosomes of P. aeruginosa, E. coli and the non-lipopolysaccharide containing Saccharomyces cerevisiae had similar protective activities. No protection was obtained with the ribonucleic acid from the E. coli phage MS 2. It is concluded that ribosomal RNA has protective activities distinct from those of LPS. 相似文献
16.
The influence of Klebsiella oxytoca and Enterobacter cloacae inoculation on dinitrogen fixation by the rice-bacteria association was examined in pots in a greenhouse. For inoculation, K. oxytoca NG13 isolated from a Japan paddy soil, E. cloacae E26 isolated from a China soil and following three strains were employed. K. oxytoca NG1389 is a mutant from NG13 and has no nitrogenase activity ( nif). K. oxytoca NG13/pMC71A and E. cloacae E26/pMC71A were produced by inserting a nif A containing K. pneumoniae plasmid (pMC71A) to NG13 and E26, respectively. These two strains were able to fix dinitrogen fixation in the presence of
ammonium, whereas nitrogenase activity of wild strains (NG13 and E26) were repressed under this condition.
Inoculation effects were tested on two rice ( Oryza sativa L.) varieties, a Indica type C5444 and a Japonica type T65. Rice seedlings were planted to nonsterilized potted soil, and
grown under flooded conditions. Upon inoculation with NG13 and E26, growth and N increment of plants particularly in T65 were
stimulated above NG1389 inoculated plants. Assay by 15N dilution and acetylene reduction techniques indicated that this N increment may be due to fixed N. Inoculation with NG13/pMC71A
and E26/pMC71A resulted in more dry weight and fixed N than those of NG13 and E26 inoculated plants.
Dr Y. Hirota died on 23 December 1986. 相似文献
17.
A bstract
Attachment of Enterobacter cloacae EcCT-50,—a biological seed protectant used to control the seed-rotting fungi, Pythium ultimum—to cotton seed was examined using conventional fluorescent microscopy (CFM), scanning electron microscopy (SEM), and laser
scanning microscopy (LSM). In sand microcosms, E. cloacae quickly attached to the seed coat, with maximum attachment, 3 to 5 h after inoculation at 24°C. In contrast, initial attachment
of non-bacterized seed by Pythium ultimum was not observed until 6 h (and not until 8 h on bacterized seeds). Comparison of the movement of E. cloacae and P. ultimum in seed exudate gradient semi-soft agar showed faster movement by the bacterium within the first 6 h, and reduction of P. ultimum hyphal and germ tube growth in the presence of the bacterium. Microscopic observation of the seed coat revealed an early,
intimate association, mediated, in part, by fimbriae, and confirmed a loose association of E. cloacae with the seed coat previously reported. Spatially, the attached E. cloacae cells were distributed over the entire surface of the seed coat, but were especially abundant in the groves and near cracks
where water imbibition and seed exudate release may occur. As the seed germinated and exposed various seed tissues, the bacterium
rapidly attached to these tissues. Attachment of the bacterium to the surface of intact germinating seeds, excised seed coat,
polystyrene, and glass was 300, 110, 51, and <1 cell field −1 3 h −1, respectively, suggesting that attachment is enhanced by seed germination. Attachment of E. cloacae to the seed coat was optimum in sands with high water concentrations, at temperatures of 18 to 30°C, and at times that corresponded
with optimum water imbibition during germination. Using several assays, attachment was shown to be enhanced by seed exudate,
and compounds such as methanol, fructose, and calcium. The results suggest that the release of certain nutrients and water
imbibition during germination may play a role in the rapid attachment to the seed by E. cloacae. The ability of E. cloacae to rapidly move and attach to the seed coat may be related to its ability to function as a biocontrol agent.
Received: 24 April 1997; Accepted 29 October 1997 相似文献
18.
In this study, a homogenous polysaccharide (FSP), with an average molecular weight of 9.08 × 10 4 Da, was isolated from Forsythia suspense and its antibacterial potential against Enterobacter cloacae producing SHV‐12 ESBL was investigated. Growth kinetics, in vitro competition and biofilm formation experiments demonstrated that SHV‐12 ESBL contributed to a fitness benefit to E cloacae strain. The antibacterial activity of FSP (2.5, 5.0 and 10.0 μg/mL) was tested against E cloacae bearing SHV‐12 ESBL gene using bacterial sensitivity, agar bioassay and agar well diffusion assays. It was found that the addition of FSP demonstrated potent antibacterial activities against this bacterial as showed by the decrease of bacterial growth and the increase of the inhibition zone diameter. Furthermore, SHV‐12 ESBL gene expression was decreased in E cloacae strain following different FSP treatment in a concentration‐dependent manner. In conclusion, these data showed that FSP exhibited potent good antibacterial activity against E cloacae producing SHV‐12 ESBL via inhibition of SHV‐12 ESBL gene expression, which may promote the development of novel natural antibacterial agents to treat infections caused by this drug‐resistant bacterial pathogen. 相似文献
19.
Both Enterobacter cloacae H478 and Klebsiella edwardsii S15 were shown to harbour a relatively large conjugative plasmid that coded for cloacin DF13-susceptibility and the production and uptake of a hydroxamate iron chelator, most probably aerobactin. Protein-blotting experiments with antiserum raised against the purified cloacin DF13/aerobactin receptor protein from Escherichia coli (Co1V-K30) revealed that the corresponding outer membrane receptor proteins of Ent. cloacae H478 and K. edwardsii S15 had apparent mol wts of 85 000 and 76000, respectively. E. coli transconjugants harbouring either the plasmid from Ent. cloacae H478 or K. edwardsii S15 expressed a cloacin DF13/aerobactin outer membrane receptor protein with a mol wt of 74000. The receptor protein encoded by the Ent. cloacae and K. edwardsii plasmids were immunologically more related to each other than to the pCo1V-K30-encoded receptor protein. 相似文献
20.
Summary A membrane-bound d(–)-lactate dehydrogenase (LDH), an important enzyme in carbon and energy metabolism in sulfate-reducing bacteria of the genus Desulfovibrio, was solubilized from the membrane fraction of Desulfovibrio desulfuricans (ATCC 7757). The enzyme was purified 84-fold to a final specific activity of 525 nmol DCPIP-reduced/min/mg protein by ammonium sulfate precipitation, chloroform extraction, gel filtration with Sephadex G-150, and hydrophobic column chromatography with N-octylamine Sepharose 4B. The enzyme eluted off a Sephacryl S-300 column as a single peak with a molecular weight of 400 000±40 000 Da. Denaturing gel electrophoresis showed it to be composed of 5 protein bands. The oxidized and dithionite reduced spectra of LDH resembles the spectra of c-type cytochromes found in Desulfovibrio species. The addition of lactate to LDH resulted in a partially reduced spectrum. The flavin/cytochrome c/non-heme iron content per 400 000 Da LDH molecular weight was found to be 11.64.5. The LDH activity was specific for d(–)-lactate and had a K
m for d(–)-lactate of 4.3×10 –4 M. The pH optimum was between 6.5 and 8.5. 相似文献
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