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PubMed1976

Variation in urease activity of endemic hospital strains of Proteus rettgeri and Providencia stuartii.

Both urease-positive and urease-negative Proteeae isolated from cross-infected patients in the same hospitals and, in three cases, from the same patients were examined for their biochemical reactions and somatic (O-) antigens. All isolates gave the same reactions in 17 biochemical tests and possessed O-antigens characteristic of Providenic O-type strains 4 or 17. Study of the isolates indicated that endemic strains are capable of undergoing variation in urease activity. In the current classification urease-positive and urease-negative strains are classified as Proteus rettgeri and Providencia stuartii, respectively. The observed variation in urease activity of nosocomial isolates of Proteeae suggests that taxonomy should be modified so that all such strains would be accommodated in a single group.

Antigens, Bacterial↗
PubMed1995

In vitro activity of subinhibitory concentrations of quinolones on urea-splitting bacteria: effect on urease activity and on cell surface hydrophobicity.

The effect of subinhibitory concentrations of ciprofloxacin, lomefloxacin, norfloxacin, ofloxacin, and sparfloxacin on urease activity and on cell surface hydrophobicity of urea-splitting bacteria was examined. Quinolones at 0.5 MICs demonstrated variable effects on bacterial-urease activity. Norfloxacin inhibited enzyme activity in Proteus vulgaris and Proteus mirabilis, while other quinolones had no effects. In Morganella morganii, sparfloxacin and ciprofloxacin enhanced urease activity, particularly at the initial phase of growth. All quinolones tested showed no marked effect on urease activity by Providencia rettgeri. Quinolones at the same concentrations induced an increase in the cell surface hydrophobicity, which was strain-dependent. There was no correlation between urease inhibition and cell surface hydrophobicity. Inhibition of urease activity by quinolones, in addition to their antibacterial activities, may prevent the progression of urinary tissue damage and stone formation.

Anti-Infective Agents↗
PubMed1995

Urease production correlates with possession of the trh gene in Vibrio parahaemolyticus strains isolated in Thailand.

A total of 489 Vibrio parahaemolyticus isolates from patients in Thailand with diarrhea was examined for the presence of thermostable direct hemolysin (TDH) and TDH-related hemolysin genes (tdh and trh, respectively), their serovars, TDH production, and urease activity. Of the strains, 81% were positive only for the tdh gene, 6% for both trh and tdh genes, and 2% for the trh gene only. Thirty-seven (8%) of the 489 isolates were positive for urease production. Of special interest, all urease-positive strains possessed the trh gene, and conversely, urease-negative strains lacked the gene, indicating that urease production by V. parahaemolyticus strains strongly correlates with the possession of the trh gene. Thus, the urease-positive phenotype of V. parahaemolyticus can be considered an indication of virulent (trh-possessing) V. parahaemolyticus strains in clinical diagnosis.

Bacterial Proteins↗
PubMed1998

Feeding diets containing high levels of milk products or cellulose decrease urease activity and ammonia production in rat intestine.

Three studies were done to determine the effect of feeding diets containing high levels of a readily fermentable carbohydrate (lactose in milk or yogurt, or pure lactose) or an undigestible, unfermentable diluent (alpha-cellulose) on urease (EC 3.5.1.5) activity and net ammonia production in the rat gastrointestinal (GI) contents. Rats (170-200 g) were fed a control diet or diets containing 55% dried milk or 55% dried yogurt, 25% lactose or 10% alpha-cellulose. Feeding diets containing milk or yogurt decreased urease activity to approximately 11% of the control value in the small intestine (on the basis of grams of collected contents or total contents), and to 50% in the large intestine (only on the basis of grams of collected contents). Feeding the diet containing 25% lactose also decreased urease activity (on the basis of grams of collected contents or total contents) to about 20% of the control value in the small intestine, but not (P > 0.05) in the large intestine. Net ammonia production rate was correlated (r2 = 0.98) with urease activity in the large intestinal contents, and the rate of ammonia production from ureolysis represented about two thirds of the total. Feeding the cellulose diet decreased (P < 0.05) both urease activity and net ammonia production in the large intestine to approximately 30% of the control value. Weights of tissue and contents of the large intestine were much higher (P < 0.01) in rats fed diets containing milk products or lactose than in the control rats, but were not affected by consumption of the cellulose diet. Results of our studies indicate that feeding diets containing high levels of milk products (lactose) or cellulose reduces urease activity and net ammonia production in the rat intestine, and thus may be beneficial for improving animal and human health.

Ammonia↗
PubMed1990

Generation of ammonia and mucosal lesion formation following hydrolysis of urea by urease in the rat stomach.

We examined the morphological changes in gastric mucosa and the generation of ammonia after exposure of the rat stomach to urea in the presence of urease, in attempts to investigate a pathophysiological role of urea, urease, and ammonia system in gastric ulcer diseases. Exposure of the stomach for 20 min to 2 ml urea (0.025-0.2%) together with urease (100 IU) induced histological damages in a concentration-related manner. Either urea or urease alone did not induce any histological change in the mucosa. Instillation of urea into the stomach generated ammonia in the presence of urease; the amount of ammonia was increased depending on the concentration of urea, and was closely associated with the severity of histological damage. The exposure of the stomach to ammonia (NH4OH: 0.01-0.1%) also produced histological damages in the gastric mucosa in a concentration-related manner. The characteristics of injury induced by 0.5-1.0% ammonia were stasis of microcirculation, disruption of the surface epithelial cells, and necrosis of the mucosa. These results demonstrated that ammonia generated from the hydrolysis of urea by urease in the stomach causes damages in the gastric mucosa.

Ammonia↗
PubMed1998

Premedication with Xylocaine spray does not lead to a false positive rapid urease test.

Rapid urease tests are used for quick identification of Helicobacter pylori during upper gastrointestinal endoscopy. Rapid urease test solutions contain urea, which in the presence of H. pylori urease, generates ammonia, which changes the test medium color to indicate a positive result. Theoretically, Xylocaine spray (ASTRA, Södertalje, Sweden), which has a basic pH value, could cause a similar positive reaction in the test medium. To determine whether patients premedicated with Xylocaine spray have a higher rate of false positive urease tests, we compared the results of a rapid urease test and histologic stains in 107 patients, 54 premedicated with Xylocaine spray and 53 premedicated with intravenous midazolam but not Xylocaine spray. There were no significant differences in test sensitivity, specificity, or predictive values between the study groups. We conclude that patients can be premedicated with Xylocaine spray without concern that the false positive rate of rapid urease tests will increase.

Anesthetics, Local↗
PubMed2004

Evaluation of a new biopsy urease test: Pronto Dry, for the diagnosis of Helicobacter pylori infection.

BACKGROUND: The gastric biopsy urease test is the most frequently used test for the diagnosis of Helicobacter pylori infection in routine gastrointestinal endoscopy practice. In Malaysia up to recently, only one commercial biopsy urease test was available: the CLO test (Ballard Medical Products, Draper, Utah, USA). Large endoscopy units use their own 'homemade' unbuffered ultra rapid urease test for diagnosis of H. pylori infection. OBJECTIVE: To compare the accuracy and reaction time of a new biopsy urease test, Pronto Dry (Medical Instruments Corporation, Solothurn, Switzerland) and the CLO test in the diagnosis of H. pylori infection. METHODS: Consecutive patients presenting with dyspepsia to the endoscopy unit, University of Malaya Medical Centre were recruited for the study. Patients who were previously treated for H. pylori infection or who had received antibiotics, proton pump inhibitors or bismuth compounds in the preceding 4 weeks were excluded. H. pylori diagnosis was made based on the ultra rapid urease test and histological examination of gastric biopsies. Four antral and four corpus biopsies were taken for this purpose from all patients. A diagnosis of H. pylori infection was made when both the ultra rapid urease test and histology were positive in either the antral or corpus biopsies. A negative diagnosis of H. pylori was made when both tests from antral and corpus biopsies were all negative. Another four antral and four corpus biopsies (two each) were taken for the Pronto Dry and CLO tests. The Pronto Dry and CLO tests were stored and performed according to the manufacturer's instruction. RESULTS: Two hundred and eight patients were recruited in the study. Eighty-six of the patients were males and 122 were females. The mean age was 46.3 years with a range of 15-82 years. The results for both the Pronto Dry and the CLO tests were completely concordant with sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of 98.1%, 100%, 100%, 98.1% and 99%, respectively. The Pronto Dry test showed a faster reaction time to positive compared with the CLO test, with 96.2% positive reaction by 30 min versus 70.8% and 100% positive reaction time by 55 min versus 83%. The colorimetric change was also more distinct with the Pronto Dry test compared with the CLO test. CONCLUSIONS: Both the Pronto Dry and the CLO tests were highly accurate for the diagnosis of H. pylori infection. The Pronto Dry test showed a quicker positive reaction time and the positive colour change was more distinct.

Adolescent↗
PubMed1989

Regulation of gene expression and cellular localization of cloned Klebsiella aerogenes (K. pneumoniae) urease.

The genes for Klebsiella aerogenes (K. pneumoniae) urease were cloned and the protein was overexpressed (up to 18% of total protein consisted of this enzyme) in several hosts. The small size of the DNA encoding urease (3.5 kb), the restriction map, and the regulation of enzyme expression directed by the recombinant plasmid are distinct from other cloned ureases. Nickel concentration did not affect urease gene expression, as demonstrated by the high levels of apoenzyme measured in cells grown in nickel-free media. However, nickel was required for urease activity. The overproducing recombinant strain was used for immunogold electron microscopic localization studies to demonstrate that urease is a cytoplasmic enzyme.

Bacterial Proteins↗
PubMed1988

The urease enzymes of Campylobacter pylori and a related bacterium.

The urease enzyme of Campylobacter pylori was studied and compared with that of a related spiral-shaped bacterium, St1, isolated from the rodent ileum. Both bacteria possessed constitutive urease enzymes with activities up to 20-70 times that of Proteus vulgaris. This activity was retained on SDS-polyacrylamide gels. A major catalytic subunit of mol. wt 300,000 was located for all (six) strains of C. pylori subjected to SDS-PAGE whereas St1 had two active forms of mol. wts 140,000 and 150,000. Western-blot analysis indicated the presence of anti-urease antibodies in the sera of patients with C. pylori-associated gastritis. The response to C. pylori urease was not strain-specific but no cross-reactivity was detected between the C. pylori enzyme and that of St1. The very high urease activity of these bacteria is likely to be important in colonisation of the host. Possession of glutamate dehydrogenase activity by both organisms suggests that one role of the urease may be to assimilate the available urea nitrogen. Modification of the local environment to facilitate long-term colonisation is another possible function. Protection from acid is unlikely to be a primary role as the natural habitat of the organism St1 is the non-acid-secreting tissue of the small intestine.

Ammonia↗
PubMed2003

Differential regulation of the Proteus mirabilis urease gene cluster by UreR and H-NS.

Proteus mirabilis, a cause of catheter-associated urinary tract infection, relies on several virulence factors to colonize the urinary tract. Among these, urease contributes to the development of urinary stones resulting from the increase in local pH due to urease-mediated hydrolysis of urea to NH(3) and CO(2). UreR, an AraC-like transcriptional activator, activates transcription of the genes encoding the urease subunits and accessory proteins (ureDABCEFG) in the presence of urea. UreR also initiates transcription of its own gene in a urea-inducible manner by binding to the intergenic region between ureR and ureD. The intergenic region contains poly(A) tracts that appear to be the target of H-NS. It has been shown that Escherichia coli and P. mirabilis H-NS acts to repress transcription of ureR in an E. coli model system. It was hypothesized that H-NS represses urease gene expression in the absence of UreR and urea by binding to the intergenic region. To demonstrate this the P. mirabilis hns gene was cloned and the 15.6 kDa H-NS was overexpressed and purified as a myc-His tail fusion. Using a gel shift assay, purified H-NS-myc-His bound preferentially to a 609 bp DNA fragment containing the entire ureR-ureD intergenic region. H-NS and UreR were able to displace each other from the ureR-ureD intergenic region. Circular permutation analysis revealed that the intergenic region is bent. Moreover, H-NS recognizes this curvature, binds the DNA fragment and induces further bending of the DNA as shown by a circular ligation assay. The effects of H-NS, urea and temperature (25 vs 37 degrees C) on urease expression were shown in E. coli containing an hns knockout and P. mirabilis where expression was increased at 37 degrees C. Increased transcription from p(ureR) was seen in the E. coli hns knockout when temperature was increased from 25 to 37 degrees C. These findings suggest H-NS and UreR differentially regulate urease in a negative and positive manner, respectively.

Bacterial Proteins↗
PubMed2003

The urease gene cluster of Vibrio parahaemolyticus does not influence the expression of the thermostable direct hemolysin (TDH) gene or the TDH-related hemolysin gene.

In order to investigate why the thermostable direct hemolysin (TDH) and the TDH-related hemolysin (TRH) of Vibrio parahaemolyticus are produced at low levels from urease-positive strains, the effect of the functional urease gene cluster of V. parahaemolyticus on the expression of the tdh and trh genes was examined. Transcriptional lacZ fusions with the tdh1, tdh2, trh1 and trh2 genes representing variants of the tdh and trh genes were integrated into the chromosome of an Escherichia coli strain and a urease-negative V. parahaemolyticus strain. The plasmid-borne urease gene cluster introduced and expressed in these constructs did not affect expression of any of the fusion genes. The amount of TDH produced from a Kanagawa phenomenon-positive V. parahaemolyticus did not change by introduction of the urease gene cluster either. It was concluded therefore that the urease gene cluster is not involved in the regulation of tdh and trh expression.

Bacterial Proteins↗
PubMed1991

In vitro inhibition of Helicobacter pylori urease: biochemical and ultrastructural analysis.

Inhibition of H. pylori urease was studied by means of electron-microscopy and electrophoretic methods using different urease inhibitors, such as acetohydroxamic-acid (AHA), L-ascorbic acid (AsA), copper ions, a combination of L-ascorbic acid with copper ions and UV light. AHA in two different concentrations and AsA at a concentration of 0.1 mg ml-1 showed incomplete inhibition of H. pylori urease activity in our electrophoretic experiments. Only membrane-bound activity was inhibited with AHA but not the activity localized within the cytoplasma as demonstrated by electron-microscopy. AsA at a concentration of 0.5 mg ml-1 and the combination of copper ions (1 microgram ml-1) with AsA completely inhibited the urease activity as demonstrated by electron-microscopy and electrophoretic experiments. Cu2+ ions in high concentrations (100 micrograms ml-1) and UV light exposure for more than 4 h induced a complete disintegration of H. pylori. Electrophoresis showed no active protein after UV light exposure of 2 h. Different urease inhibitors tested in this study showed dose-dependent inhibitory effects on H. pylori urease in vitro.

Ascorbic Acid↗
PubMed1995

UreR activates transcription at multiple promoters within the plasmid-encoded urease locus of the Enterobacteriaceae.

Urease activity is produced by members of the family Enterobacteriaceae that contain the plasmid-encoded urease locus only when urea is present in the growth medium. The plasmid-encoded urease locus contains seven tandem urease structural and accessory genes (ureDABCEFG). Previously we showed that transcription of the first gene in this cluster, ureD, is initiated at a urea-dependent promoter (ureDp). Expression from ureDp requires the product of ureR, which is transcribed divergently from the plasmid-encoded ureDABCEFG. From DNA sequence analysis, UreR is predicted to be a 34 kDa protein with identity to the AraC family of transcriptional activators. In this report we demonstrate that there are two additional urea and UreR-dependent promoters within the plasmid-encoded urease locus: ureRp and ureGp. A low-level constitutive promoter was also identified upstream of ureE (ureEp). Three major mRNA transcripts were induced when urea was present in the growth medium: a transcript containing ureDABCEF, a transcript corresponding to ureG, and a transcript corresponding to ureR. These results indicate that expression of each of the plasmid-encoded urease genes is transcriptionally regulated in response to urea and suggest that there is autogenous regulation of ureR. Therefore UreR is one of three AraC family members described thus far that are positively auto-regulated.

Blotting, Northern↗
PubMed1977

Electron microscopy of negatively stained jackbean urease at three levels of quaternary structure, and comparison with hydrodynamic studies.

Electron microscopy, with sodium phosphotungstate as negative stain, has been carried out on purified jackbean urease prepared at three levels of quaternary structure: (a) A1 urease, Mr = 240 000, S20,W = 11.5 S (b) alpha urease, Mr = 480 000, S20,w = 18.3 S (c) polymers of alpha urease above the tetramer stage. The compatibility of the images from level to level leaves no doubt that the enzyme itself is being visualized, and the following geometry is suggested by electron microscopy: A1 molecules are cyclic trimers, which pair up in eclipsed position across a 1-nm cleft to form the hexameric alpha, which displays D3 (or 32) symmetry of a trigonal prism. Polymers consist of alpha molecules aligned with their clefts coplanar and an angle of 120 degrees between each triplet of 3-fold axes. These features correspond reasonably well with sedimentation and electrophoretic studies of the solvated enzyme, which have indicated a hemispherical A1, a spherical alpha, and string-of-beads polymers. Sedimentation constants of the urease polymers up through the pentamer level were found to be compatible with the rosette, straight-chain, and zig-zag forms seen in the electron microscope, and with the suggested protomer arrangement in A1 and alpha urease.

Macromolecular Substances↗
PubMed1993

Structural change of jack bean urease induced by addition of surfactants studied with synchrotron-radiation small-angle X-ray scattering.

Both the quaternary and subunit structures of jack bean urease in solutions with ionic and nonionic surfactants have been studied by small-angle X-ray scattering using a synchrotron-radiation source. The effects of those surfactants on the enzyme activity of urease have also been investigated in the same kind of solvent systems as those used for the scattering experiments. The present results show that the quaternary structure of urease in solution is fairly elongate and that by the relatively minor binding of SDS (SDS/protein = 0.23/1) the native urease molecule is dissociated into six identical subunits with nearly spherical structures. In addition the enzyme activity of urease was mostly retained under every solvent condition investigated, indicating that the subunit found in the present scattering experiments is the fundamental monomeric unit for both the quaternary-structure formation and enzyme function of urease.

Fabaceae↗
PubMed1996

Cytotoxin and urease activities of Helicobacter pylori isolates from Japanese patients with atrophic gastritis or duodenal ulcer.

The vacuolating cytotoxin and urease secreted by Helicobacter pylori are thought to be virulent factors. Because vacuolation is potentiated by the presence of ammonium ion, which is produced by urease in vitro, it is of interest to examine whether cytotoxin and urease work reciprocally in the development of atrophic gastritis or duodenal ulcer. In the present study, patients (all H. pylori-positive) were divided into four groups: mild atrophic gastritis (group 1; nine patients), severe atrophic gastritis (group 2; 36 patients), duodenal ulcer with mild atrophic gastritis (group 3; 19 patients) and duodenal ulcer with severe atrophic gastritis (group 4; 12 patients). Cytotoxin production and urease activity of H. pylori isolated from these patients were analysed. Cytotoxin production was observed in four of nine (44.4%), 28 of 36 (77.8%), 11 of 19 (57.9%) and eight of 12 (66.7%) isolates from groups 1, 2, 3 and 4, respectively. Cytotoxin-producing H. pylori isolates were found significantly more in patients with severe atrophy than in patients with mild atrophy (P = 0.048). The mean of relative activity of cytotoxin in H. pylori isolate was 1.6 +/- 2.3, 7.9 +/- 7.4, 5.8 +/- 6.0 and 9.0 +/- 9.1 in groups 1, 2, 3 and 4, respectively. Helicobacter pylori isolates from severe atrophy or duodenal ulcer patients in groups 2 or 4 possessed significantly higher activity than those from non-ulcer patients in group 1 (P = 0.017 and 0.030, respectively). The mean of urease activity was 8.6 +/- 4.6, 10.0 +/- 5.9, 10.0 +/- 8.5 and 11.2 +/- 7.7 IU/mg in groups 1, 2, 3 and 4, respectively. These differences indicated no statistical significance. In each H. pylori isolate, the production of cytotoxin and urease were independent, which indicated that there was no reciprocal effect between them in vivo. Thus, cytotoxin-producing H. pylori isolates were more prevalent in patients with severe atrophic gastritis and the cytotoxin activities of H. pylori isolates from the patients with severe atrophic gastritis or duodenal ulcer were much higher than those from the patients with mild atrophic gastritis, which suggested that vacuolating cytotoxin may be a disease-inducing factor.

Bacterial Proteins↗
PubMed2006

Mucosal immunization with a urease B DNA vaccine induces innate and cellular immune responses against Helicobacter pylori.

BACKGROUND: Helicobacter pylori is recognized as a major risk factor for recurrent gastroduodenal inflammatory diseases and gastric adenocarcinoma. The high prevalence of H. pylori infection worldwide, the risks of side-effects from antibiotic therapy, and increasing resistance to antibiotics are the main primers for the development of improved H. pylori vaccines. The antigenic potential of its urease enzyme, a critical virulence factor required for colonization of the gastric mucosa, has been demonstrated in animal and human studies. An important but controversial issue in H. pylori vaccine studies is the type of immune response required to control infection. A new approach in H. pylori vaccinology is the administration of DNA vaccines, which has included heat-shock protein and catalase DNA vaccines. MATERIALS AND METHODS: The H. pylori urease subunit B construct or vector alone was administered to mice via the intranasal route. Spleens and stomachs were examined on day 0 and weeks 3, 6, and 12 after immunization. Proliferation of spleen cells was assessed using the carboxyfluorescein diacetate succinimidyl ester-based flow cytometry assay and cytokine secretion from cultured spleen cells was detected by ELISA, after stimulation with the urease subunit B recombinant antigen. Total RNA was isolated from stomach and spleen tissue and the expression of beta-defensin and cytokine genes was monitored by reverse transcription followed by polymerase chain reaction (RT-PCR). Immunized mice were challenged with H. pylori and bacterial DNA quantified by TaqMan PCR. RESULTS: The urease B subunit DNA vaccine increased INF-gamma secretion and splenocyte proliferation without inducing adverse effects in the spleen. Increase in gastric beta-defensin 1 and marked induction in local IL-10 : IFN-gamma ratio up to 12 weeks post-immunization suggest a potential role for local innate immune responses in protection at the site of infection. Although significant bacterial reduction in the stomachs of urease B subunit DNA-immunized mice was observed, intermediate reduction was also noted in the vector group. Increased defensin expression and adjuvant effects of the cytosine preceding guanosine motifs may contribute to this phenomenon. Our data confirm that cytosine preceding guanosine motifs, even without coadministration with antigen, can reduce extracellular bacterial load. CONCLUSIONS: In this study, a DNA construct encoding the urease B subunit was assessed for its immune profile and its ability to reduce bacterial colonization in the murine stomach. Our studies suggest that local innate immune responses may play a greater role than previously supposed in limiting H. pylori colonization in the gastric mucosa.

Animals↗
PubMed1998

Validation of a serologic test for the diagnosis of Helicobacter pylori infection and the immune response to urease and CagA in children.

OBJECTIVE: Little is known about Helicobacter pylori infections and the immune response to urease and CagA in pediatric populations. Our aims were: 1) to validate serological assays for antibodies against whole cell extract, CagA, and urease of H. pylori; 2) to examine their role in diagnosis of infection in children with recurrent abdominal pain (RAP); and 3) to examine the antibody responses to CagA and urease in children. METHODS: An enzyme-linked immunosorbent assay (ELISA) for diagnosis of H. pylori infection using whole cell extracts was validated in 50 children with biopsy-confirmed infection. The IgG and IgA antibody responses against recombinant CagA and urease were compared by ELISA in 82 children with RAP and in 246 age- and sex-matched healthy children. RESULTS: The whole-cell extract ELISA had a sensitivity of 85 % and specificity of 87%. Children with RAP were more infected with H. pylori than were healthy control subjects; however, IgG and IgA CagA seropositivity was lower among those with RAP than among asymptomatic children (34% and 23% vs 76% and 55%, respectively; p < 0.0001). In both groups of children, the immune response to urease was low. CONCLUSION: A serodiagnosis of H. pylori infection using native strains was developed. The difference in the immune response between children with RAP and control subjects suggests that RAP occurs during the acute phase of H. pylori infection. Our results also suggest that urease is a poor immunogen.

Abdominal Pain↗