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PubMedDate not supplied

Use of urease-bromothymol blue-agar method for large-scale testing of urine on grain and seeds.

The current AOAC method (963.28) for large-scale (50 g) testing of urine on grain is based on the reaction of sodium in urine with magnesium uranyl acetate. Detection of sodium suggests that urine is present and that a test for urea is appropriate. Urea is detected with urease-bromothymol blue-paper and is confirmed through its reaction with xanthydrol to form dixanthylurea crystals, which are detected microscopically. The initial nonspecific test for sodium can be influenced by the presence of salt or other sodium compounds. Furthermore, the magnesium uranyl acetate spray used in Method 963.28 potentially exposes the analyst to the aerosol of a volatile, toxic uranium compound. Excess reagents and analyzed test portions must be disposed of as radioactive waste. In addition, Method 963.28 requires several steps to determine the presence of urea. The alternative AOAC method (972.41) tests for the presence of urea from urine on individual seeds. Urea is enzymatically decomposed to ammonia and carbon dioxide by urease. Liberated ammonia shifts the pH, changing the color of the indicator in the agar from yellow to blue. This study adapts Method 972.41 to larger test samples. Up to 25 g grains and seeds are sprayed with urease test agar instead of being individually immersed in the urease test agar. The modified method was used to analyze urea on seeds and grains of 24 plants from 4 families. The method has a limit of detection of one seed contaminated with 1 microgram urea.

Aerosols↗
PubMed1996

[Urease test, microbiologic tests, histologic and serologic tests for the evaluation of Helicobacter pylori infections in persons with peptic ulcer and gastritis].

The presence of Helicobacter pylori in the gastric mucosa of 150 patients with gastric, duodenal ulcer and endoscopic gastritis was investigated by rapid urease-based test, culture, histological examination and serology. Overall, 78% were positive for Helicobacter pylori by rapid urease-based test, 69.3% by culture, 53.7% by histological staining and 93.2% by serology (p < 0.001). The frequency of Helicobacter pylori was higher in patients with gastric or duodenal ulcer compared with those with endoscopic gastritis and those after gastric resection. However, the differences were significant only with reference to rapid urease-based test, (p < 0.02). The EIA (Roche) serological test had the highest sensitivity and the lowest specificity. This indicates the seed for serological kit to be independently evaluated on the population to be studied. The rapid urease-based test was simple, rapid and inexpensive, and it was more sensitive and equally specific compared with culture and Giemsa stain.

Adolescent↗
PubMed1997

Relationship between state of a thermosensitive matrix and the activity of urease immobilized in it.

The effect of temperature on kinetic and equilibrium parameters of urea hydrolysis catalyzed with urease immobilized into a thermosensitive poly-N-isopropylacrylamide gel was studied. The temperature behavior of the gel-urease system is different from similar systems. After a decrease in the enzyme activity above the critical temperature, the maximal rate of the enzymatic reaction and gel swelling ratio begin to increase. Urea hydrolysis catalyzed with immobilized urease and shrinking-swelling of the thermosensitive urease-containing gel depend on each other. Under collapse, gel swelling increases due to the enzymatic reaction. The rate of the enzymatic reaction no longer follows Michaelis-Menten kinetics, and the dependence of the reaction rate on substrate concentration becomes more complicated.

Acrylamides↗
PubMed1998

Chemotaxis of Helicobacter pylori: a urease-independent response.

Helicobacter pylori has adapted to a very specialized niche; namely, the highly acidic, viscous, and somewhat anaerobic gastric mucus of humans. The enzyme urease is essential for colonization of the stomach, as it provides protection against gastric acidity. A spiral morphology as well as sheathed flagella have been claimed to give the bacteria an advantage in colonizing mucus. Wild type strain of H. pylori and its isogenic urease-negative mutant showed a chemotactic response to urea, flurofamide (a potent urease inhibitor), sodium ion, and bicarbonate ion. These chemotactic responses are also observed in the viscous environment. Thus, it appears that H. pylori has chemotactic movement that is independent of urease activity. The chemotactic response was inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), a potent H+ pump inhibitor, but not by Na+ pump-inhibiting amiloride, suggesting this response is forced by H+-driven flagellar movement. Since urea and sodium bicarbonate are secreted from the gastric epithelial surface, this chemotactic response may contribute to the colonization by H. pylori and the persistence of its infection.

Chemotaxis↗
PubMed2004

Structural characterization of the nickel-binding properties of Bacillus pasteurii urease accessory protein (Ure)E in solution.

Urease activation is critical to the virulence of many human and animal pathogens. Urease possesses multiple, nickel-containing active sites, and UreE, the only nickel-binding protein among the urease accessory proteins, activates urease by transporting nickel ions. We performed NMR experiments to investigate the solution structure and the nickel-binding properties of Bacillus pasteurii (Bp) UreE. The secondary structures and global folds of BpUreE were determined for its metal-free and nickel-bound forms. The results indicated that no major structural change of BpUreE arises from the nickel binding. In addition to the previously identified nickel-binding site (Gly(97)-Cys(103)), the C-terminal tail region (Lys(141)-His(147)) was confirmed for the first time to be involved in the nickel binding. The C-terminally conserved sequence ((144)GHQH(147)) was confirmed to have an inherent nickel-binding ability. Nickel addition to 1.6 mm subunit, a concentration where BpUreE predominantly forms a tetramer upon the nickel binding, induced a biphasic spectral change consistent with binding of up to at least three nickel ions per tetrameric unit. In contrast, nickel addition to 0.1 mm subunit, a concentration at which the protein is primarily a dimer, caused a monophasic spectral change consistent with more than 1 equivalent per dimeric unit. Combined with the equilibrium dialysis results, which indicated 2.5 nickel equivalents binding per dimer at a micromolar protein concentration, the nickel-binding stoichiometry of BpUreE at a physiological concentration could be three nickel ions per dimer. Altogether, the present results provide the first detailed structural data concerning the nickel-binding properties of intact, wild-type BpUreE in solution.

Amino Acid Sequence↗
PubMed1970

Urease color test medium U-9 for the detection and identification of "T" mycoplasms in clinical material.

A urease color test fluid medium (U-9) for the detection and identification of T (T-strain) mycoplasmas in clinical material is described which is sensitive and specific for this group of mycoplasmas. The medium was prepared from commercially available components and contained 95% half-strength, tryptic digest broth (pH 5.5), 4% unheated horse serum, 0.05% highest-purity urea, 0.001% sodium phenolsulfonphthalein, and 1,000 units of potassium penicillin G per ml. The final reaction of medium U-9 was pH 6.0. The overall agreement (positive and negative) between urease reactions in U-9 urease color test medium and culture findings in a standard agar primary culture system among 686 clinical specimens was 98.1%. The disagreement consisted of 13 false-positive urease reactions which were recognized visually as false-positive reactions due to other microorganisms. For specimens from the female genitourinary tract, the inclusion of 2.5 mug of amphotericin B (Fungizone) per ml of medium U-9 is recommended for the suppression of growth of Candida species and filamentous fungi.

Bacteriological Techniques↗
PubMed1976

Effects of immobilization on the kinetics of enzyme-catalyzed reactions. II. Urease in a packed-column differential reactor system.

Urease from Jack bean was immobilized on nonporous glass beads by covalent bonding and its kinetics were studied in a packed-column differential reactor. To facilitate comparison, the urease was immobilized by both diazo and glutaraldehyde coupling. The kinetic properties of immobilized urease were similar to those of the soluble enzyme and different immobilization methods did not appreciably alter the kinetic properties. The affects of three different amino acid activators appear to follow predictions obtained from a relatively simple competitive model, except at very low substrate levels.

Enzyme Activation↗
PubMed1994

Transport and hydrolysis of urea in a reactor-separator combining an anion-exchange membrane and immobilized urease.

A membrane reactor-separator, in which an anion-exchange membrane and a urease-immobilized poly(vinyl alcohol) (PVA) membrane were clamped together to separate the feed solution and the stripping solution of a dialysis cell, was constructed. The urea in the feed solution passed through the anion-exchange membrane, water film, and then was hydrolyzed to ammonium carbamate in the urease-immobilized PVA membrane. The experimental results showed that no ammonium ion was found in the feed solution under either phosphate or citrate buffer systems at 0.05-0.2 mol dm-3 and pH 6-9, and various initial concentrations of urea in the feed solution (20-200 mmol dm-3). This indicates that the water film between two membranes allows the carbamate ions to decompose into ammonium and carbonate ions completely before entering the anion-exchange membrane. The device therefore can be used for the removal of urea from feed solution, while preventing the backflow of ammonium ions from the stripping solution or water film into feed solution. It has significant potential in the development of a wearable or portable artificial kidney. The properties of the urease-immobilized PVA membrane were examined. A kinetic model describing the transport-reaction behavior of urea in the membrane reactor-separator was developed, and the optimum values of the reactor parameters were obtained.

Biological Transport↗
PubMed2003

Macromolecular assembly of Helicobacter pylori urease investigated by mass spectrometry.

The supramolecular assembly of Helicobacter pylori urease was studied by nanoflow electrospray ionization orthogonal time-of-flight mass spectrometry. The measured molecular mass of the urease complex of 1.06 MDa corresponds to a dodecameric (alphabeta)(12) assembly of urease alpha (26 kDa) and beta (61 kDa) subunits. The dodecamer disassembles readily into (alphabeta)(3) subunits in solution and under controlled collisional-induced dissociation in the gas phase. This is in strong support of an ((alphabeta)(3))(4) architecture consistent with the recently published x-ray structure. In vitro, the alpha and beta subunits are capable of re-assembling to (alphabeta)(3), but not further to the dodecameric complex.

Amino Acid Sequence↗
PubMed1995

Urease activity in the crystalline state.

Crystalline Klebsiella aerogenes urease was found to have less than 0.05% of the activity observed for the soluble enzyme under standard assay conditions. Li2SO4, present in the crystal storage buffer at 2 M concentration, was shown to inhibit soluble urease by a mixed inhibition mechanism (Ki's of 0.38 +/- 0.05 M for the free enzyme and 0.13 +/- 0.02 M for the enzyme-urea complex). However, the activity of crystals was less than 0.5% of the expected value, suggesting that salt inhibition does not account for the near absence of crystalline activity. Dissolution of crystals resulted in approximately 43% recovery of the soluble enzyme activity, demonstrating that protein denaturation during crystal growth does not cause the dramatic diminishment in the catalytic rate. Finally, crushed crystals exhibited only a three-fold increase in activity over that of intact crystals, indicating that the rate of substrate diffusion into the crystals does not significantly limit the enzyme activity. We conclude that urease is effectively inactive in this crystal form, possibly due to conformational restrictions associated with a lid covering the active site, and propose that the small amounts of activity observed arise from limited enzyme activity at the crystal surfaces or trace levels of enzyme dissolution into the crystal storage buffer.

Binding Sites↗
PubMed1990

Molecular modeling studies on the urease active site and the enzyme-catalyzed urea hydrolysis.

These studies are an attempt to gain better insight into the pharmacophore requirements of urease. On the basis of published information on this enzyme (EXAFS, amino acid sequence, essential groups at the active site) a hypothetical nickel-tripeptide complex, as preliminary substitute for the urease active site was modeled using computer-aided molecular modeling techniques. The results suggest two alternative docking modes of urea and reaction intermediates, corresponding to two different reaction mechanisms. Both binding modes are compatible with the docking of known potent inhibitors such as selected hydroxamic acids and phosphorodiamides. The results can be used to help in the design of new potential inhibitors of urease.

Binding Sites↗
PubMed1993

Evaluation of rapid urease test for detection of Helicobacter pylori in gastric biopsy specimens.

Using 80 gastric biopsy specimens from patients with various gastroduodenal diseases, the isolation rate of H. pylori and urease activity of the biopsy specimens were examined. The sensitivity and specificity of the rapid urease test was 84.6% and 75.9%, respectively, for the cultivation of H. pylori. The mean number of H. pylori detected in urease-positive and negative biopsy specimens was 10(6.1 +/- 0.9) and 10(4.5 +/- 1.2) cfu/g specimens, respectively.

Adult↗
PubMed1979

Urease stones.

Urinary stones form as a consequence of urinary supersaturation. Supersaturation occurs as a result of elevated concentrations of urinary solutes. Dietary, metabolic, endocrine, hereditary, and infectious processes alter urinary solute concentrations. Struvite (MgNH4PO. 6H2O) and carbonate-apatite [Ca10(PO4)6CO3] stones form in urine that becomes supersaturated as a by-product of the hydrolysis of urea by the bacterial enzyme urease. Urease-induced stones manifest primarily as branched renal calculi and as bladder calculi. Conventional therapy has usually consisted of surgical removal of the stone combined with a short course of antimicrobial therapy. Such treatment is curative in about 50% of cases. Recurrent stone formation and progressive pyelonephritis occur in those who are not cured. Adjunctive medical treatment with acetohydroxamic acid or hydroxyurea lessens the risk of calculogenesis and decreases growth of residual stones in patients who are not cured by conventional therapy. Patients with urea-splitting urinary infection and renal stones have a major life-threatening disease. The morbidity and expense that result from this disease are great. Long-term (perhaps lifetime) chemotherapy with antimicrobial agents and/or urease-inhibiting drugs combined with judicious and expert surgical intervention can be expected to significantly improve the plight of these unfortunate patients.

Adult↗
PubMed1986

The effects of glycosaminoglycans, pyrophosphate and allopurinol treatment on urease-induced crystallization in vitro.

Previous studies have found that human urine inhibits urease-induced crystallization in synthetic urine. To identify the inhibitory components the effects of chondroitin sulphate, heparin and pyrophosphate on urease-induced crystallization were studied and the inhibitory capacity of human urine collected during and before/after allopurinol treatment compared. None of the substances studied nor allopurinol treatment was found to influence the urease-induced crystallization. These findings do not support the idea that glycosaminoglycans or pyrophosphate are responsible for the inhibitory effect detected in human urine.

Allopurinol↗
PubMed1988

Adherence of urease-induced crystals to rat bladder epithelium.

Apart from urine supersaturation with respect to struvite and calcium phosphate caused by urease-producing microorganisms, retention of formed crystals in the urinary tract is necessary for the formation of infection stones. This study was performed to investigate the role of the mucous coat lining the urothelium in the adhesion of urease-induced crystals. Removal of this glycosaminoglycan-containing layer from rat bladders increased the adherence of struvite and calcium phosphate crystals 5-6 times compared to that in intact rat bladders. Heparin completely restored the antiadherence capacity while chondroitin sulphate had a very weak restorative effect and human urine had no restorative effect. These findings support the view that the mucous coat is of importance in preventing retention of urease-induced crystals.

Animals↗
PubMed1990

Effects of serum, albumin and immunoglobulins on urease-induced crystallization in urine.

The effects of serum, albumin and gammaglobulins on urease-induced crystallization have been studied in synthetic and in human urine. Serum and the studied proteins increased urease enzymatic activity in synthetic urine. In human urine only serum had this effect. In synthetic urine, the proteins and serum markedly decreased the precipitation attached to glass surfaces, while the intraluminal precipitation was increased. In human urine, similar but weaker effects on the precipitation were found for serum and albumin. These findings suggest that the proteins studied, in the concentrations in which they are present in human urine, have profound effects on urease-induced crystallization and may be physiological crystallization inhibitors.

Crystallization↗
PubMed1978

Reversion and interallelic complementation at four urease loci in Neurospora crassa.

Analysis of heat stability of urease in extracts of 24 revertants, six for each of four ure loci, revealed that at least one revertant for each locus had a heat stability about one-third that of wild type. Similar results were obtained with urease formed by interallelic complementation at the ure-2 and ure-4 loci, but interallelic complementation at the ure-1 and ure-3 loci produced insufficient urease activity for analysis. The data are interpreted to suggest, as a tentative model, a structural function for each of the four ure loci.

Alleles↗
PubMed1976

Inactivation of allantoinase from Pseudomonas aeruginosa by a subunit of urease.

Cell-free extracts prepared from Pseudomonas aeruginosa cells, cultured in a medium containing allantoin as sole nsource of carbon, nitrogen and energy and harvested in the stationary phase, contain an enzymicly inactive allantoinase-inhibitor complex. Pure inhibitor was isolated by dissociation of this complex followed by gelfiltration. The inhibitor had a molecular weight of about 5500 daltons. Association between inhibitor and allantoinase was demonstrated by gelfiltration and by polyacrylamide gel-electrophoresis. The inhibitor was unstable in the absence of 1 M urea and the inactivation was accompanied by aggregate formation and appearance of urease activity. The inhibitor was also isolated from cells containing urease but no allantoinase. It was concluded that the inhibitor is a subunit of urease. Inhibitors isolated from P. aeruginosa and P. acidovorans cells were active against both allantoinase from P. aeruginosa and allantoinase from P. acidovorans.

Allantoin↗