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PubMed2004

Urease expression by Cryptococcus neoformans promotes microvascular sequestration, thereby enhancing central nervous system invasion.

Our objective was to determine the role of the cryptococcal virulence factor urease in pulmonary-to-central nervous system, dissemination, invasion, and growth. C. neoformans H99, the urease knockout strain (ure1) derived from H99, and the urease restored strain ure1+URE1-1 were used for the studies. The absence of cryptococcal urease (ure1infection) resulted in significant protection from the high mortality observed in H99-infected mice. All H99-infected mice had extremely high cryptococcal loads in their brains at the time of death, whereas only two of six animals that died of ure1 infection had detectable C. neoformans in the brain. Histological analysis of the blood-to-brain invasion by C. neoformans H99 demonstrated wedging of the yeasts in small capillaries, altered structure of microvessel walls, formation of mucoid cysts initiated in the proximity of damaged microcapillaries, and the absence of an inflammatory response. Direct inoculation of H99, ure1, and ure1+URE1-1 into the brain demonstrated that urease was not required to grow in the brain. However, the dissemination patterns in the brain, spleen, and other organs after intravenous inoculation indicated that cryptococcal urease contributes to the central nervous system invasion by enhancing yeast sequestration within microcapillary beds (such as within the brain) during hematogenous spread, thereby facilitating blood-to-brain invasion by C. neoformans.

Animals↗

PubMed1998

Ecabet sodium, a locally acting antiulcer drug, inhibits urease activity of Helicobacter pylori.

In order to clarify the mechanism of the anti-Helicobacter pylori action of ecabet sodium (ecabet), a locally acting antiulcer drug, we evaluated the effects of ecabet on H. pylori urease activity in vitro. H. pylori was cultured and a crude preparation of urease was made. Urea-dependent survival of H. pylori at acid pH was significantly inhibited by ecabet. The urease activity of intact cells and a crude enzyme preparation from H. pylori had two pH optima: pH 4.5-5.0 and 8.0. Ecabet (1-4 mg/ml) concentration dependently inhibited the urease activity of both preparations at pH 5.0, but there was no inhibition at pH 8.0. The enzyme activity was inhibited by ecabet gradually and was not restored by dilution, in contrast to the inhibition elicited by benzohydroxamic acid, a specific and reversible urease inhibitor. These results suggest that irreversible inhibition of H. pylori urease activity contributes to the anti-H. pylori action of ecabet.

Abietanes↗

PubMed1998

Influence of pH on metabolism and urease activity of Helicobacter pylori.

BACKGROUND & AIMS: The metabolic and urease responses of Helicobacter pylori to variations in gastric acidity are unknown. The aim of this study was to determine effects of changes of environmental pH on metabolism, urease activity, and survival of H. pylori in an unbuffered environment. METHODS: Bacterial metabolism and urease activity were determined by measuring pH changes in perfused microphysiometer chambers over a pH range from 2.5 to 9.0 with or without urea and survival by restoration of metabolism at pH 7.4. RESULTS: Glucose metabolism by acid-adapted H. pylori occurred at a perfusion pH between 3.5 and 8.6 and was highest between 7.4 and 8.2. Metabolism was irreversibly inhibited at pH <3.5 or >8.6. In the presence of 2.5 mmol/L urea, the chamber pH increased to about 6.2 during perfusion between pH 5.5 and 4.0. At pH 4.0 and below, urease activity increased several-fold without change of chamber pH. Urea in the perfusate enabled retention of metabolism after acid exposure but was toxic at pH 7.4. CONCLUSIONS: The metabolic range of acid-adapted H. pylori is between an environmental pH of 3.5 and 8.6. Extracellular pH-regulated internal urease activity allows metabolism in the pH range between 4.0 and 2. 5 by maintaining periplasmic pH at 6.2. The organism is an acid-tolerant neutralophile due to internal urease activity.

Ammonium Chloride↗

PubMed1991

Urease and the acidosis of urinary intestinal diversion.

Previous investigators have suggested that urinary tract infections with urea-splitting organisms may be a primary etiologic factor in the acidosis which is seen after urinary diversion. This study employs a model in which small intestinal segments are perfused with an artificial urine solution over a three hour period. Urease is then added in order to determine its effect on acid-base balance and net intestinal electrolyte transport. Urease created no significant increase in acid load (delta HCO3- = -7.5 +/- 2.2 for controls vs. -8.7 +/- 2.9 for urease group), but did increase the osmolality of the intestinal contents and resulted in a 24% increase in free water loss (p = .037). Analysis of sodium and chloride movement following the addition of urease to the perfusate suggests that both ammonium and bicarbonate are absorbed by the intestinal segment. Thus any acidosis resulting from increased ammonium absorption following the addition of urease appears to be offset by concomitant bicarbonate absorption. The azotemia of urinary diversion appears to be primarily the result of urea absorption, partially the result of ammonium absorption, and is not significantly increased by urease.

Acid-Base Equilibrium↗

PubMed1997

Preparation of polymeric urease discs by an electron beam irradiation technique.

A preparation method of immobilized urease discs by an electron beam irradiation technique was developed, and the relationship between enzyme activity and preparation conditions was investigated. The immobilized urease disc was a thin circular film (200 microm, 5 mm phi) that is useful for biomedical applications. The activity of urease irradiated with 1 Mrad at room temperature was protected by the presence of cysteine. The activity of the immobilized urease discs was studied as a function of monomer concentration (80-90%) and the thicker disc gave a high activity. The durability of the immobilized urease discs gave a high activity. The durability of the immobilized urease discs was evaluated by repeated batch enzyme reactions, and a high activity yield (80-85%) was obtained.

Cysteine↗

PubMedDate not supplied

Prospective evaluation of a new rapid urease test (Pronto Dry) for the diagnosis of Helicobacter pylori infection.

AIM: Rapid urease tests are commonly used to establish the diagnosis of Helicobacter pylori infection during upper endoscopy. The aim of this study was to evaluate the performance of a new rapid urease test (Pronto Dry) compared with histology as the gold standard. METHODS: Six gastric biopsies (three in the antrum and three in the fundus) were performed in 113 consecutive patients. Eighteen patients were later excluded from analysis because they did not fulfil the inclusion criteria. Four biopsies were examined by two experienced pathologists blinded to the rapid urease tests. Two biopsies (one from antrum and one from the fundus) were pooled for the rapid urease test which was read by the endoscopist 5 and 30 minutes later using the color scale (yellow, pink, orange, dark pink, fuchsia) provided by the manufacturer. RESULTS: According to the histology findings 32 of the 95 patients retained for analysis (33.7%) were positive for Helicobacter pylori. Considering that a positive test was indicated by the dark pink or fuchsia colors, sensitivity and specificity of Pronto Dry were 62.5% and 98.4% at 5 minutes and 84.4% and 98.4% at 30 minutes respectively. Twenty-one of the 28 positive rapid urease tests (75%) were already positive at 5 minutes. CONCLUSION: Considering positive tests are indicated solely by the two darkest colors on the color scale, the performance of Pronto Dry is similar to that of other rapid urease tests. The rapid results provided by Pronto Dry in routine practice would seem to provide obvious advantages.

Aged↗

PubMed2002

Persistence of immobilised and total urease and phosphatase activities in a soil amended with organic wastes.

This paper reports on the persistence of total and immobilised enzyme activities (urease and phosphatase) in a soil amended with organic wastes containing high levels of total-urease and phosphatase activity. Fresh organic materials showed the highest values for both total-enzymatic activities. The addition of organic waste to soil increased both total-enzymatic activities in the soil, which, after 360 days, showed values above those of the control. Immobilised enzymes were also higher in the fresh wastes than in the soil with compost, while the specific enzymatic activity levels (enzymatic activity per unit of carbon) were similar. The immobilised urease activity was greater in the amended soil than in the control. At the beginning of the incubation period, the immobilised urease activity was significantly higher in the soil amended with fresh organic wastes than with compost. However, this activity decreased with incubation, whilst the compost-immobilised urease activity increased with time. The effect of organic amendment on immobilised phosphatase activity was similar to that shown by immobilised urease but less pronounced. The persistence of both enzymes was significantly higher in the soil amended with compost than in that amended with fresh materials.

Enzymes, Immobilized↗

PubMed1999

A new proposal for urease mechanism based on the crystal structures of the native and inhibited enzyme from Bacillus pasteurii: why urea hydrolysis costs two nickels.

BACKGROUND: Urease catalyzes the hydrolysis of urea, the final step of organic nitrogen mineralization, using a bimetallic nickel centre. The role of the active site metal ions and amino acid residues has not been elucidated to date. Many pathologies are associated with the activity of ureolytic bacteria, and the efficiency of soil nitrogen fertilization with urea is severely decreased by urease activity. Therefore, the development of urease inhibitors would lead to a reduction of environmental pollution, to enhanced efficiency of nitrogen uptake by plants, and to improved therapeutic strategies for treatment of infections due to ureolytic bacteria. Structure-based design of urease inhibitors would require knowledge of the enzyme mechanism at the molecular level. RESULTS: The structures of native and inhibited urease from Bacillus pasteurii have been determined at a resolution of 2.0 A by synchrotron X-ray cryogenic crystallography. In the native enzyme, the coordination sphere of each of the two nickel ions is completed by a water molecule and a bridging hydroxide. A fourth water molecule completes a tetrahedral cluster of solvent molecules. The enzyme crystallized in the presence of phenylphosphorodiamidate contains the tetrahedral transition-state analogue diamidophosphoric acid, bound to the two nickel ions in an unprecedented mode. Comparison of the native and inhibited structures reveals two distinct conformations of the flap lining the active-site cavity. CONCLUSIONS: The mode of binding of the inhibitor, and a comparison between the native and inhibited urease structures, indicate a novel mechanism for enzymatic urea hydrolysis which reconciles the available structural and biochemical data.

Bacillus↗

PubMed2003

A negative rapid urease test is unreliable for exclusion of Helicobacter pylori infection during acute phase of ulcer bleeding. A prospective case control study.

BACKGROUND: The reliability of the rapid urease test has not been proven in patients with peptic ulcer bleeding. Some studies show bad diagnostic results with the rapid urease test for gastrointestinal bleeding. AIMS: To evaluate the efficacy of the rapid urease test in patients with bleeding gastric or duodenal ulcers. PATIENTS AND METHODS: A total of 96 patients with acute peptic ulcer bleeding without proton pump inhibitor or antibiotic therapy within the last 14 days before bleeding were included into the study. During index endoscopy, specimens for histological and rapid urease test were obtained from the antrum and corpus mucosa of the stomach. Patients were also investigated by the 13C-urea breath test. Diagnostic quality parameters were calculated with the histology and the 13C-urea breath test as reference and compared with a matched control group with uncomplicated ulcers. RESULTS: The sensitivity of the rapid urease test was 80% and the specificity 100% compared to histology and 13C-urea breath test. The negative predictive value was 75%. These values were statistically significantly different from those of the control group (sensitivity 96%, specificity 100%, negative predictive value 88%). CONCLUSION: The exclusive use of the rapid urease test cannot be recommended in patients with peptic ulcer bleeding.

Adult↗

PubMed1997

Structures of Cys319 variants and acetohydroxamate-inhibited Klebsiella aerogenes urease.

Cys319 is located on a mobile flap covering the active site of Klebsiella aerogenes urease but does not play an essential role in catalysis. Four urease variants altered at position C319 range from having high activity (C319A) to no measurable activity (C319Y), indicating Cys is not required at this position, but its presence is highly influential [Martin, P. R., & Hausinger, R. P. (1992) J. Biol. Chem. 267, 20024-20027]. Here, we present 2.0 A resolution crystal structures of C319A, C319S, C319D, and C319Y proteins and the C319A variant inhibited by acetohydroxamic acid. These structures show changes in the hydration of the active site nickel ions and in the position and flexibility of the active site flap. The C319Y protein exhibits an alternate conformation of the flap, explaining its lack of activity. The changes in hydration and conformation suggest that there are suboptimal protein-solvent and protein-protein interactions in the empty urease active site which contribute to urease catalysis. Specifically, we hypothesize that the suboptimal interactions may provide a significant source of substrate binding energy, and such hidden energy may be a common phenomenon for enzymes that contain mobile active site loops and undergo an induced fit. The acetohydroxamic acid-bound structure reveals a chelate interaction similar to those seen in other metalloenzymes and in a small molecule nickel complex. The inhibitor binding mode supports the proposed mode of urea binding. We complement these structural studies with extended functional studies of C319A urease to show that it has enhanced stability and resistance to inhibition by buffers containing nickel ions. The near wild-type activity and enhanced stability of the C319A variant make it useful for further studies of urease structure-function relationships.

Amino Acid Sequence↗

PubMedDate not supplied

Highly multilayered urease decomposes highly concentrated urea.

Urease was immobilized at a density of 1.2 g of urease per gram of a matrix via ion-exchange binding of urease to an anion-exchange polymer chain grafted onto a pore surface of a porous hollow-fiber membrane and subsequent cross-linking of urease with transglutaminase. Urea was hydrolyzed during the permeation of a urea solution, the concentration of which ranged from 2 to 8 M, through the pores of the resultant membrane with a thickness of approximately 1 mm. Quantitative hydrolysis of 4 M urea was achieved at a permeation rate lower than 1 mL/h, i.e., a residence time longer than 5.1 min, at ambient temperature. This performance is ascribed to convective transport of urea through the pores rimmed by the urease-immobilized polymer chains at a high density. Urease was denatured in the presence of urea at concentrations higher than 6 M while hydrolyzing urea.

Adsorption↗

PubMed1976

A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe.

1. A convenient method of preparation of jack-bean urease (EC3.5.1.5) involving covalent chromatography by thiol-disulphide interchange is described. 2. Urease thus prepared has specific activity comparable with the highest value yet reported (44.5 +/- 1.47 kat/kg, Km = 3.32 +/- 0.05 mM; kcat. = 2.15 X 10(4) +/- 0.05 X 10(4)s-1 at pH7.0 and 38 degrees C). 3. Titration of the urease thiol groups with 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) and application of the method of Tsou Chen-Lu [(1962) Sci. Sin. 11, 1535-1558] suggests that the urease molecule (assumed to have mol.wt. 483000 and epsilon280 = 2.84 X 10(5) litre-mol-1-cm-1) contains 24 inessential thiol groups of relatively high reactivity (class-I), six 'essential' thiol groups of low reactivity (class-II) and 54 buried thiol groups (class-III) which are exposed in 6M-guanidinium chloride. 4. The reaction of the class-I thiol groups with 2-Py-S-S-2-Py was studied in the pH range 6-11 at 25 degrees C(I = 0.1 mol/l) by stopped-flow spectrophotometry, and the analogous reaction of the class-II thiol groups by conventional spectrophotometry. 5. The class-I thiol groups consist of at least two sub-classes whose reactions with 2-Py-S-S-2-Py are characterized by (a) pKa = 9.1, k = 1.56 X 10(4)M-1-s-1 and (b) pKa = 8.1, k = 8.05 X 10(2)M-1-s-1 respectively. The reaction of the class-II thiol groups is characterized by pKa = 9.15 and k = 1.60 X 10(2)M-1-s-1. 6. At pH values 7-8 the class-I thiol groups consist of approx. 50% class-Ia groups and 50% class-Ib groups. The ratio class Ia/class Ib decreases an or equal to approx. 9.5, and at high pH the class-I thiol groups consist of at most 25% class-Ia groups and at least 75% class-Ib groups. 7. The reactivity of the class-II thiol groups towards 2-Py-S-S-2-Py is insensitive to the nature of the group used to block the class-I thiols. 8. All the 'essential' thiol groups in urease appear to be eeactive only as uncomplicated thiolate ions. The implications of this for the active-centre chemistry of urease relative to that of the thiol proteinases are discussed.

2,2'-Dipyridyl↗

PubMed2000

Identification of the urease operon in Helicobacter pylori and its control by mRNA decay in response to pH.

We investigated the transcription of the urease gene cluster ureABIEFGH in Helicobacter pylori to determine the regulation of gene expression of the highly produced enzyme urease. Northern blot hybridization analysis demonstrated that cells of the wild-type strain grown in an ordinary broth had transcripts of ureAB, ureABI, ureI, ureIE' and ure'FGH, but cells of a ureI-disrupted mutant had only the ureAB transcript. When the wild-type cells were exposed to pH 8 for 30 min, very little mRNA was detected. However, when exposed to pH 6, a large amount of the ureIE" transcript, which was longer than the ureIE' transcript, together with the additional transcripts ureABIEFGH and ure'EFGH were detected. Rifampicin addition experiments demonstrated that urease mRNAs, and the ureIE' transcripts in particular, are more stable at pH 5.5 than at pH 7. In accord with these results, urease activity in the crude cell extract of the pH 5.5 culture was twice as much as that of the pH 7 culture, although the amounts of UreA and UreB detected by immunoblot analysis were similar. The transcription start point of ureI was identified by primer extension using a ureA promoter-deleted mutant, and a consensus sequence of RpoD-RNA polymerase was found in the ureI promoter. The 3' end of the ureIE" mRNA, determined using S1 nuclease mapping, revealed that the transcript is able to cover the majority of the ureE open reading frame (ORF) that might be sufficient for UreE activity. Based on the above results, we conclude that the urease gene cluster of H. pylori consists of two operons, ureAB and ureIEFGH, and that primary transcripts of the latter as well as the read-through transcript, ureABIEFGH, are cleaved to produce several species of mRNA. It has been suggested that the ureIEFGH operon is regulated post-transcriptionally by mRNA decay in response to environmental pH. We are tempted to speculate that the ureE" transcript present in acidic pH may contribute to produce an active product that can proceed the nickel incorporation to the active centre, the final step of urease biosynthesis.

Amino Acid Sequence↗

PubMed1999

New inhibitors of Helicobacter pylori urease holoenzyme selected from phage-displayed peptide libraries.

Urease is an important virulence factor for Helicobacter pylori and is critical for bacterial colonization of the human gastric mucosa. Specific inhibition of urease activity has been proposed as a possible strategy to fight this bacteria which infects billions of individual throughout the world and can lead to severe pathological conditions in a limited number of cases. We have selected peptides which specifically bind and inhibit H. pylori urease from libraries of random peptides displayed on filamentous phage in the context of pIII coat protein. Screening of a highly diverse 25-mer combinatorial library and two newly constructed random 6-mer peptide libraries on solid phase H. pylori urease holoenzyme allowed the identification of two peptides, 24-mer TFLPQPRCSALLRYLSEDGVIVPS and 6-mer YDFYWW that can bind and inhibit the activity of urease purified from H. pylori. These two peptides were chemically synthesized and their inhibition constants (Ki) were found to be 47 microM for the 24-mer and 30 microM for the 6-mer peptide. Both peptides specifically inhibited the activity of H. pylori urease but not that of Bacillus pasteurii.

Amino Acid Sequence↗

PubMed2001

Rapid urease test: effect of preimmersion of biopsy forceps in formalin.

OBJECTIVE: The rapid urease test is widely used at endoscopy to determine the presence of Helicobacter pylori. The effect of immersion of the biopsy forceps in formalin before performance of a rapid urease test is uncertain. The aim of this prospective study was to determine whether previous immersion of the biopsy forceps in formalin influences rapid urease test results. METHODS: Two hundred fifty-three consecutive patients undergoing upper GI endoscopy with a clinical indication for rapid urease test had two tests with two different antral mucosa biopsies. The former was performed with a sterile biopsy forceps, the latter with a sterile biopsy forceps that was previously immersed in 10% buffered formalin. In 132 patients the CLOtest was used and in 121 patients the Jatrox-H.P. test was used. The kappa concordance test was used for comparisons. RESULTS: In the 132 patients in which the CLOtest was used, both were positive in 76 cases, both negative in 27, and discordant results were seen in 3 cases (2.27%), kappa 0.953 (range 0.901-1.000, p < 0.001). In the 121 patients in which the Jatrox test was used, both were positive in 89, both negative in 27 and there were 5 (4.13%) discordant cases, kappa 0.880 (0.793-0.984, p < 0.001). There were no differences in positivity rates at 30 minutes, 1 hour, and 3 hours between the two rapid urease test kits. CONCLUSION: Preimmersion of the biopsy forceps in formalin does not adversely affect the rapid urease test.

Biopsy↗

PubMed1977

Urease activity in the digestive tract of the chick and its role in the utilisation of urea as a source of non-amino nitrogen.

Urease activity was found in caecal contents (about 10 mg urea metabolised/g h) and crop contents (about 0-5 mg urea metabolised/g h):there was very low activity in the contents of the colon but none in the rest of the digestive tract. 2. The urease activity of the crop contents was not bacterial in origin but the soyabean meal contained in the diet was found to have comparable activity. 3. Diets low in non-essential nitrogen and based on soyabean, fish meal or fish meal plus 0-2% jack bean urease, did not support higher growth rates when supplemented with urea. 4. The livers of chicks fed on the diet containing fish meal, urea and urease had significantly higher concentrations of free non-essential amino acids than those of chicks fed on the same diet but with urease excluded This suggested that dietary urease affects the availability of ammonia for the synthesis of non-essential amino acids but not for growth.

Amino Acids, Essential↗

PubMed2002

Inhibition of jack bean urease by 1,4-benzoquinone and 2,5-dimethyl-1,4-benzoquinone. Evaluation of the inhibition mechanism.

1,4-benzoquinone (BQ) and 2,5-dimethyl-1,4-benzoquinone (DMBQ) were studied as inhibitors of jack bean urease in 50 mM phosphate buffer, pH 7.0. The mechanisms of inhibition were evaluated by progress curves studies and steady-state approach to data achieved by preincubation of the enzyme with the inhibitor. The obtained reaction progress curves were time-dependent and characteristic of slow-binding inhibition. The effects of different concentrations of BQ and DMBQ on the initial and steady-state velocities as well as the apparent first-order velocity constants obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. The rapid formation of an initial BQ-urease complex with an inhibition constant of Ki = 0.031 mM was followed by a slow isomerization into the final BQ-urease complex with the overall inhibition constant of Ki* = 4.5 x 10(-5) mM. The respective inhibition constants for DMBQ were Ki = 0.42 mM, Ki* = 1.2 x 10(-3) mM. The rate constants of the inhibitor-urease isomerization indicated that forward processes were rapid in contrast to slow reverse reactions. The overall inhibition constants obtained by the steady-state analysis were found to be 5.1 x 10(-5) mM for BQ and 0.98 x 10(-3) mM for DMBQ. BQ was found to be a much stronger inhibitor of urease than DMBQ. A test, based on reaction with L-cysteine, confirmed the essential role of the sulfhydryl group in the inhibition of urease by BQ and DMBQ.

Benzoquinones↗

PubMed1998

Immunization of mice with urease vaccine affords protection against Helicobacter pylori infection in the absence of antibodies and is mediated by MHC class II-restricted responses.

We examined the roles of cell- and antibody-mediated immunity in urease vaccine-induced protection against Helicobacter pylori infection. Normal and knockout mice deficient in major histocompatibility complex (MHC) class I, MHC class II, or B cell responses were mucosally immunized with urease plus Escherichia coli heat-labile enterotoxin (LT), or parenterally immunized with urease plus aluminum hydroxide or a glycolipid adjuvant, challenged with H. pylori strain X47-2AL, and H. pylori organisms and leukocyte infiltration in the gastric mucosa quantified. In an adjuvant/route study in normal mice, there was a direct correlation between the level of protection and the density of T cells recruited to the gastric mucosa. In knockout studies, oral immunization with urease plus LT protected MHC class I knockout mice [beta2-microglobulin (-/-)] but not MHC class II knockout mice [I-Ab (-/-)]. In B cell knockout mice [microMT (-/-)], vaccine-induced protection was equivalent to that observed in immunized wild-type (+/+) mice; no IgA+ cells were detected in the stomach, but levels of CD4(+) cells equivalent to those in the wild-type strain (+/+) were seen. These studies indicate that protection of mice against H. pylori infection by immunization with the urease antigen is dependent on MHC class II-restricted, cell-mediated mechanisms, and antibody responses to urease are not required for protection.

Adjuvants, Immunologic↗