Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “UREASE”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 541 records · Page 30Linked to original sources
PubMed1995

Inhibition of Helicobacter pylori urease by phenyl phosphorodiamidates: mechanism of action.

Helicobacter pylori urease is a nickel-containing enzyme that hydrolyzes urea to bicarbonate and ammonia. Andrews et al. (J. Am. Chem. Soc. 1986, 108, 7124) have shown that amides and esters of phosphoric acid are slow, tight-binding inhibitors of urease isolated from jack bean. We show that 4-substituted phenyl phosphorodiamidates (4-R-PhOP(=O)(NH2)2) are slow-binding inhibitors of H. pylori urease with no evidence of kinetic saturation. Their second-order rates of inhibition ki are strongly correlated with phenol pKa (e.g. R = NO2, ki = 2.5 x 10(5) M-1s-1; R = OMe, ki = 1.2 x 10(4) M-1s-1). The Bronsted beta for inhibition is 0.4, similar to that of model system SN2(P) reactions. Based on these observations, we suggest that urease inhibition is covalent but reversible, involving a common phosphoacyl enzyme intermediate.

Helicobacter pylori↗
PubMed2004

The use of Fourier transform infrared spectroscopy to assay for urease from Pseudomonas aeruginosa and Canavalia ensiformis.

A novel assay method was investigated for urease (EC 3.5.1.5) from Pseudomonas aeruginosa and Canavalia ensiformis by Fourier transform infrared spectroscopy. This enzyme catalyzed the hydrolysis of urea in phosphate buffer in deuterium oxide ((2)H(2)O). The intensities of the bicarbonate bands maxima at 1625 and 1365 cm(-1) and of the amide I band at 1605 cm(-1) were measured as a function of time to study the kinetics of urea hydrolysis. The extinction coefficients epsilon of urea and bicarbonate were determined to be 0.72, 0.48, and 0.56 mM(-1)cm(-1) at 1625, 1605, and 1365 cm(-1), respectively. The initial velocity is proportional to the enzyme concentration by using the ureases from both C.ensiformis and P. aeruginosa. The kinetic constants (V(max), K(m), and K(cat)) determined by Lineweaver-Burk plot were 532.2 U mg(-1) protein, 6.4mM, and 806.36 s(-1), respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on glutamate dehydrogenase in aqueous media. Therefore, this spectroscopic method is highly suited to assay for urease activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of urease activity.

Canavalia↗
PubMed2006

Combined effect of heavy metals and polycyclic aromatic hydrocarbons on urease activity in soil.

Actions and interactions between heavy metals (HM)-cadmium, zinc, lead, and polycyclic aromatic hydrocarbons (PAHs)-phenanthrene, fluoranthene, benzo(a)pyrene toward soil urease activity were studied after 7, 14, 21, and 28 days of exposure under controlled conditions. The experimental approach was based on the uniform design. Ten different contamination conditions were studied simultaneously, with 10 concentration levels for each pollutant. Data treatment was essentially based on the multiple regression technique. The results showed that Zn interacted more easily with PAHs than Pb or Cd. On the first 7 days of incubation, zinc alone reduced the urease activity more significantly than any other pollutants and no significant interactions between PAH and HM were observed. From 14 to 21 days of incubation, the interaction between Zn and benzo(a)pyrene decreased the soil urease activity. At 14 days, the interaction between Zn and phenanthrene was antagonistic (less than additive), while at 21 days it was synergistic (more than additive). At 28 days, the interaction between phenanthrene and fluoranthene was synergistic. This study indicated that the combined effect of PAH and HM on soil urease activity depends largely on the incubation time. Uniform design appears to be a good method for investigating the combined effect of PAH and HM.

Drug Combinations↗
PubMed2005

A catalytic antibody heavy chain HpU-2 degrading its epitope peptide and H. pylori urease.

The HpU-2 monoclonal antibody (mAb) raised against Helicobacter pylori urease mainly recognized the alpha-subunit of the urease. On the other hand, the heavy chain of HpU-2 mAb (HpU-2-H) isolated from the parent mAb recognized both the alpha- and beta-subunit, in which the beta-subunit was recognized more strongly than the beta-subunit. HpU-2-H cleaved a peptide, SVELIDIGGNRRIFGFNALVD, which is the epitope sequence recognized by HpU-2 mAb, showing a double-phase reaction profile at 25 degrees C in a phosphate buffer. After an induction time of 24h, the cleavage of the peptide was initiated by HpU-2-H at a high rate and it was completed at 80 h of incubation. By mass spectroscopy, two main fragmented peptides, SVELIDIGGNRR and SVELIDIGGNRRIFG, were identified. In addition, many small peptide fragments were produced by successive cleavage of the fragmented peptides. Cleavage tests for H. pylori urease by HpU-2-H revealed that the beta-subunit of the urease was cleaved first and completely decomposed at 20 h of incubation. Cleavage of the alpha-subunit started after the complete decomposition of the beta-subunit. These cleavage results were in good agreement with the immunological features of HpU-2-H. The irrelevant proteins, BSA and HSA, were hardly cleaved by HpU-2-H.

Amino Acid Sequence↗
PubMed2004

A plasmid immunization construct encoding urease B of Helicobacter pylori induces an antigen-specific antibody response and upregulates the expression of beta-defensins and IL-10 in the stomachs of immunized mice.

The objectives of this study were to investigate the efficacy of a prototype DNA immunization construct encoding the urease B subunit enzyme of Helicobacter pylori (H. pylori) for inducing adaptive and innate immune responses in mice immunized via intramuscular or subcutaneous routes and to further explore the adjuvant effects of the CpG motifs in the vector. Antibody, cytokine, and beta-defensin profiles were assessed in the stomachs of immunized animals: experiments were terminated 3 months after immunization because there was a significant increase in the anti-H. pylori urease B antibody response at Week 6 in mice immunized with the urease B construct. A long lasting expression of IL-10 mRNA was noted. Furthermore, a marked and sustained increase in the mRNA expression of beta-defensins was also observed, particularly beta1. This study demonstrates that an H. pylori urease B DNA construct can induce innate as well as adaptive immune responses in the stomachs of immunized mice. Upregulation of beta-defensin gene expression followed immunization and we believe that this is the first report of a DNA vaccine inducing innate anti-microbial responses. Such complex molecular interactions that modulate both innate and adaptive immune responses may be of critical importance in the control of mucosal pathogens, such as H. pylori.

Animals↗
PubMed2000

A prospective evaluation of new rapid urease tests before and after eradication treatment of Helicobacter pylori, in comparison with histology, culture and 13C-urea breath test.

BACKGROUND: The rapid urease test is a simple and cost-effective method to detect Helicobacter pylori in biopsy specimens. The aim of this study was to evaluate the accuracy of two new rapid urease tests, Helicocheck and PyloriTek, before and after eradication. METHODS: A total of 278 patients, including 115 patients who had not undergone eradication of H pylori and 163 patients after eradication treatment, were enrolled. Eight biopsy specimens were taken from both the antrum and the body of the stomach for histology, culture, and two rapid urease tests. Assessment of H pylori infection was determined by the combination of histology, culture, and (13)C-urea breath test. RESULTS: Overall sensitivity, specificity, and positive and negative predictive values of the Helicocheck before eradication were 91.0%, 100%, 100%, and 62.5%; PyloriTek, 92.0%, 100%, 100%, and 65.2%. Those of Helicocheck after eradication were, respectively, 60. 5%, 99.2%, 95.8%, and 89.2%; PyloriTek, 60.5%, 99.2%, 95.8%, and 89. 2%. For the Helicocheck, determination of the infection status of H pylori by biopsies from the gastric body had a significantly higher sensitivity than antral biopsies. After eradication, the combination of 1 antral biopsy and 1 biopsy from the body was not effective enough to improve the overall sensitivity. CONCLUSIONS: Helicocheck and PyloriTek have equally satisfactory overall sensitivity before eradication treatment. However, the sensitivity of these rapid urease tests was lower after eradication than before eradication.

Biopsy↗
PubMed1996

Prospective comparison of commercially available rapid urease tests for the diagnosis of Helicobacter pylori.

BACKGROUND: Rapid urease testing is the initial endoscopic test of choice for the diagnosis of Helicobacter pylori. Determination of the relative diagnostic yields and times to a positive test for the different rapid urease tests is important for endoscopists. We compared three commercially available tests using histologic examination and culture as a gold standard. METHODS: Patients undergoing upper endoscopy had six biopsy specimens taken from the antrum and six from the body with a large-channel biopsy forceps. Each set of six specimens was divided as follows: one each for CLOtest, Hpfast, and Pyloritek rapid urease tests; one for culture; and two for histologic examination (H&E, Genta). All tests were read every 15 minutes for 1 hour; the final reading for Pyloritek was at 1 hour. CLOtest and Hpfast were also read at 4 hours and 24 hours. RESULTS: One hundred seventy-three sets of biopsy specimens from 87 patients were evaluated; 98 (57%) of the 173 sets were positive for H. pylori by histologic examination and/or culture. The mean and median times to a positive test were significantly less for Pyloritek (0.5 +/- 0.02 hour and 0.5 hour) than for CLOtest (2.0 +/- 0.6 hour and 0.75 hour) or Hpfast (2.2 +/- 0.6 hour and 0.5 hour). The sensitivities at the final readings were similar among the three tests (CLOtest: 93%; Hpfast: 88%; Pyloritek: 89%), but sensitivities at 1 hour were significantly better for Pyloritek (89%) than for CLOtest (71%) or Hpfast (66%). At 4 hours, sensitivities for CLOtest and Hpfast improved significantly and were not significantly different from those of Pyloritek. Specificities were 99% to 100% at all times for all three tests. CONCLUSION: The three rapid urease tests, CLOtest, Hpfast, and Pyloritek, provide comparable results, with sensitivities around 90% and specificities around 100%. The Pyloritek becomes positive more rapidly than the CLOtest or Hpfast. If a reading is desired within 1 hour, the Pyloritek provides a greater sensitivity than the CLOtest or Hpfast without any sacrifice in specificity.

Bacteriological Techniques↗
PubMed1996

The influence of size or number of biopsies on rapid urease test results: a prospective evaluation.

BACKGROUND: The optimal number or size of endoscopic biopsies for use in rapid urease testing has not been established. Postulating that increasing the amount of tissue sampled would improve diagnostic yield and hasten development of a positive test, we compared urease testing with one regular biopsy, two regular biopsies, and one "jumbo" forceps biopsy. METHODS: One hundred fifty patients undergoing endoscopy had three sets of prepyloric biopsies placed in a CLOtest: one regular forceps biopsy, two regular forceps biopsy, and one large-channel jumbo forceps biopsy. Biopsies were then taken for two independent histologic examinations. Disagreements were resolved by another examiner. RESULTS: Eighty-nine patients (59%) had Helicobacter pylori infection by histology; interobserver agreement was 90% with kappa = 0.78. The mean time to a positive test was 5.3 +/- 0.9 hours for one regular biopsy, 3.2 +/- 0.7 hours for two regular biopsies, and 3.8 +/- 0.8 hours for one jumbo biopsy (p < 0.01 for two regular, one jumbo vs. one regular biopsy). Compared to one regular biopsy, the urease test was positive at least 30 minutes earlier in 56% of the patients with two regular biopsies and 54% with one jumbo biopsy. Sensitivities for one regular versus two regular biopsies were 1 hour, 19% versus 33% (p = 0.059); 2 hours, 38% versus 49% (p = 0.17); 3 hours, 48% versus 60% (p = 0.18); and 24 hours, 75% versus 79% (p > 0.20). CONCLUSIONS: Doubling the amount of tissue in the CLOtest hastens the development of a positive test by approximately 1 1/2 to 2 hours; tests become positive at least 30 minutes earlier in over 50% of the patients. Low cost, ease, and excellent specificity make the rapid urease test a valuable diagnostic tool. Nevertheless, if used as a "rapid" diagnostic test (read within 3 hours of biopsy), it is associated with a false negative rate of approximately 40%.

Adult↗
PubMed1998

Effect of blood on rapid urease testing of gastric mucosal biopsy specimens.

BACKGROUND: The effect of blood on rapid urease tests is uncertain. We assessed the effect of soaking gastric biopsy specimens in blood on the results of both agar gel (CLOtest) and strip (Pyloritek) rapid urease tests. METHODS: One hundred patients undergoing endoscopy had four adjacent biopsy specimens taken from normal appearing mucosa in the antrum. Two biopsies were soaked in blood for 1 minute; one specimen was placed on a CLOtest and one on a Pyloritek. The other two biopsy specimens were placed on CLOtest and Pyloritek without soaking in blood. The same process was performed with four adjacent biopsy specimens from the gastric body. CLOtests were read at 1, 4, and 24 hours; Pyloritek results were read at 1 hour. RESULTS: The number of positive tests for the blood-soaked and standard biopsy specimens were comparable at all times for both rapid urease tests. Discordant results between the blood-soaked and standard specimens were seen in 17 of 400 test comparisons (4%): in 8 of these only the blood-soaked specimen was positive, and in 9 only the standard specimen was positive. CONCLUSIONS: "Contamination" of biopsy specimens with blood does not alter rapid urease test results.

Biopsy↗
PubMed1986

The effects of urease in undiluted human urine.

Undiluted human urine and synthetic urine were inoculated with urease. No inhibitory activity against urease enzymatic activity could be detected in human urine. The urease-induced crystallization of both calcium phosphate and magnesium ammonium phosphate differed markedly, however, between the individuals studied, and it was less pronounced in human urine than in synthetic urine. This supports the observation made in experiments using diluted urine that human urine possesses an inhibitory activity against urease-induced crystallization and suggests that it has a large interindividual variation.

Adult↗
PubMed1988

Purification and characterization of urease from Ureaplasma urealyticum.

The urease from Ureaplasma urealyticum strain T 960 was isolated by the use of affinity chromatography, hydrophobic chromatography and gel filtration. The enzyme was purified by a factor of 155. The urease appeared as a single band of molecular weight (MW) 75,000 using reducing conditions in SDS-polyacrylamide gel electrophoresis. By gel filtration the native MW was determined to be 150,000. Isoelectric focusing showed the presence of two closely migrating enzyme species with a pI of pH 5.1-5.2. These findings show multiple forms of the urease and that these forms are composed of subunits. The electrophoresis experiments also indicate that this enzyme is a major component of the cytoplasm of U. urealyticum. The Km of the purified enzyme was 4.5 mM urea and the specific activity was 33530 mumoles NH3 x min-1 x mg-1. The optimum pH was pH 7-7.5. The urease activity was inhibited by flurofamide, acetohydroxamic acid, N-ethylmaleimide and p-chloromercuribenzoate but not by iodoacetate.

Benzamides↗
PubMed1999

Safety and immunogenicity of phoP/phoQ-deleted Salmonella typhi expressing Helicobacter pylori urease in adult volunteers.

Salmonella typhi Ty800, deleted for the Salmonella phoP/phoQ virulence regulon has been shown to be a safe and immunogenic single dose oral typhoid fever vaccine in volunteers. This promising vaccine strain was modified to constitutively express a heterologous protein of Gram negative bacterial origin, Helicobacter pylori urease subunits A and B, yielding S. typhi strain Ty1033. Seven volunteers received single oral doses of > or = 10(10) colony forming units of Ty1033; an eighth volunteer received two doses 3 months apart. Side effects were similar to those observed previously in volunteers who received the unmodified vector Ty800. All volunteers had strong mucosal immune responses to vaccination as measured by increases in IgA-secreting cells in peripheral blood directed against S. typhi antigens. Seven of eight volunteers had convincing seroconversion as measured by increases in serum IgG directed against S. typhi flagella and lipopolysaccharide antigens by ELISA. No volunteer had detectable mucosal or humoral immune responses to the urease antigen after immunization with single doses of Ty1033. A subset of three volunteers received an oral booster vaccination consisting of recombinant purified H. pylori urease A/B and E. coli heat labile toxin adjuvant 15 days after immunization with Ty1033. None of three had detectable humoral or mucosal immune responses to urease. Expression of a stable immunogenic protein in an appropriately attenuated S. typhi vector did not engender detectable mucosal or systemic antibody responses; additional work will be needed to define variables important for immunogenicity of heterologous antigens carried by live S. typhi vectors in humans.

Adult↗
PubMed2000

Purification of surface-associated urease from Helicobacter pylori.

Helicobacter pylori colonizes the human gastric mucosa and produces large amounts of urease. The enzyme was extracted from the bacteria by distilled water and purified by gel-permeation (Sephacryl S-300), anion-exchange chromatography (Mono Q) and a second gel-permeation (Superdex 200). Urease enzyme activity was detected with a spectrophotometic assay based on phenol red. The optimal pH for anion-exchange was 6.9. The recovery of urease was 55-75%, purity 93-98% and the overall protein recovery 0.8-1.4%. The urease in the final extract still had enzymatic activity and showed the typical subunits of Mr 66000 and Mr 30000 when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Blotting, Western↗
PubMed2001

Biocolloids with ordered urease multilayer shells as enzymatic reactors.

The preparation of biocolloids with organized enzyme-containing multilayer shells for exploitation as colloidal enzymatic nanoreactors is described. Urease multilayers were assembled onto submicrometer-sized polystyrene spheres by the sequential adsorption of urease and polyelectrolyte, in a predetermined order, utilizing electrostatic interactions for layer growth. The catalytic activity of the biocolloids increased proportionally with the number of urease layers deposited on the particles, demonstrating that biocolloid particles with tailored enzymatic activities can be produced. It was further found that precoating the latex spheres with nanoparticles (40-nm silica or 12-nm magnetite) enhanced both the stability (with respect to adsorption) and enzymatic activity of the urease multilayers. The presence of the magnetite nanoparticle coating also provided a magnetic function that allowed the biocolloids to be easily and rapidly separated with a permanent magnet. The fabrication of such colloids opens new avenues for the application of bioparticles and represents a promising route for the creation of complex catalytic particles.

Colloids↗
PubMed2006

Catalyzed decomposition of urea. Molecular dynamics simulations of the binding of urea to urease.

We present the results of molecular dynamics simulations on the urea/urease system. The starting structure was prepared from the 2.0 A crystal structure of Benini et al. [(1999) Struct. Folding Des. 7, 205-216] of DAP-inhibited urease (PDB code ), and the trimeric structure (2479 residues) resulted in 180K atoms after solvation by water. The force field parameters were derived using the bonded model approach described by Hoops et al. [(1991) J. Am. Chem. Soc. 113, 8262-8270]. Three different systems were analyzed, each one modeling a different protonation pattern for the His320 and His219 residues. In each case, the three monomers of urease have been analyzed separately. The time-averaged structures observed in the three monomers suggest that urease could follow two different competitive mechanisms. A "protein-assisted proton transfer" mechanism points to Asp221 as crucial for catalysis. An "Asp-mediated proton transfer" involves the transfer of a proton from the bridging OH to an NH2 moiety of urea, assisted by Asp360 in the active site. The impact of the simulation results on our understanding of urease catalysis is discussed in detail.

Bacillus↗
PubMed2002

Advantages of using non-isothermal bioreactors in agricultural waste water treatment by means of immobilized urease. Study on the influence of spacer length and immobilization method.

The behavior of three different catalytic membranes, obtained by immobilizing urease on nylon sheets chemically grafted with methyl methacrylate, was studied in a bioreactor operating under isothermal and non-isothermal conditions. Membrane activation was carried out by condensation or acyl azide reaction, and spacers of different lengths, such as hexamethylendiamine or hydrazine, were used. Under isothermal conditions, the activities of the catalytic membranes and soluble urease were characterized as a function of pH, temperature, and urea concentration. Both enzyme forms showed the same optimum pH, whereas the optimum temperature was lower for the immobilized enzymes. The spacer length appeared to determine broader pH- and temperature-activity profiles for the urease derivatives. The apparent K(m) values of the insoluble urease were dependent on membrane type and were higher than those of the soluble counterpart, thus indicating an affinity loss for urea. Under non-isothermal conditions, all membranes exhibited an increase of percentage activity proportional to the applied temperature difference and decreasing with the increase of urea concentrations. A decrease of the apparent K(m) was also observed. These results suggest that substrate diffusion limitations due to the immobilization process can be overcome in the presence of temperature gradients. In addition, the remarkable reduction of the production times supports the use of non-isothermal bioreactors for the treatment of urea-polluted waste waters.

Agriculture↗
PubMed2002

A method for estimation of urease activity in gastric mucosa biopsy specimens and Helicobacter pylori cell suspensions.

A method for measuring urease activity in biopsy specimens and Helicobacter pylori cultures from these specimens is proposed. The method is based on measurement (with a portable pH-meter) of the rate of pH changes in a reaction mixture consisting of buffer, substrate (urea), and biopsy specimen or bacterial cells. This method revealed that urease activity of biopsy specimens correlated with that of H. pylori suspension in the same experiment. High urease activity was found in biopsy specimens containing the greatest number of Helicobacter cells; only one of 14 specimens free of H. pylori cells showed no urease activity. Introduction of this method into clinical practice will help to evaluate the contribution of H. pylori to the pathological process.

Adult↗
PubMed1998

Comparison of two rapid urease tests for detection of Helicobacter pylori infection.

Rapid urease tests are widely used at endoscopy to determine the presence of Helicobacter pylori infection. In this prospective study, we compared the accuracy of two rapid urease tests, CLOtest and PyloriTek, using histology as the gold standard. Histologic staining was performed using both H&E and Giemsa, and all slides were reviewed by a single pathologist who was blinded to the results of the rapid urease tests and endoscopic findings. One hundred two patients were enrolled; their mean age was 59 years (range 16 to 95 years), and there were 45 males and 57 females. Histology confirmed the presence of H. pylori infection in 39% of patients. The proportions of false positives for CLOtest (8.0%) and PyloriTek measured at 1 hr (29.0%) were significantly different (Z = 2.90, P = 0.0038). No significant difference was seen between the proportions of false negatives. We conclude that the clinical usefulness of PyloriTek urease test is limited by its lack of specificity.

Adolescent↗