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PubMed2002

Invertase and urease activities in the carotenogenic yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma).

Invertase and urease are enzyme entities highly associated with the cells of the astaxanthin-producer yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) during any stage of its cell growth cycle. In this study cellobiose was a more efficient carbon source than sucrose or its hexose counterparts for invertase expression. Extensive ultrasonication or abrasion with glass pearls were required in order to promote enzyme release. In contrast to the yeast whose growth declines above 27 degrees C, the released enzymes displayed a higher optimum temperature range when assayed in vitro. Isoforms from both enzymes could be resolved either by FPLC on DEAE-Sepharose or by an affinity approach on immobilized Concanavalin. The zymogram for invertase showed a pI somewhat less acidic than that of the similar enzyme from S. cerevisiae.

Cell Division↗
PubMed2002

Rapid and sensitive method for prenatal diagnosis of propionic acidemia using stable isotope dilution gas chromatography-mass spectrometry and urease pretreatment.

Propionic acidemia is one of the most frequent inborn errors of metabolism caused by a deficiency of propionyl-CoA carboxylase. Methylcitric acid, a key indicator of this disorder, is increased in amniotic fluid when a fetus is affected. Therefore, the direct chemical analysis of cell-free amniotic fluid for methylcitric acid, using stable isotope dilution gas chromatography-mass spectrometry, was carried out for the prenatal diagnosis of propionic acidemia. We developed a simple, highly sensitive, and accurate method for quantitation of this polar methylcitric acid in amniotic fluids by applying a simplified urease pretreatment which we devised earlier for urine. As the recovery of methylcitric acid from amniotic fluid was as high as 91% with a coefficient of variation lower than 3% in this procedure, only 0.02 ml of sample was required for the analysis of the affected fetus. This new procedure takes 1 h for sample pretreatment, including derivatization, and 15 min for GC-MS measurement and provides final results within 1.5 h.

Amniotic Fluid↗
PubMed1994

Molecular typing of Helicobacter pylori isolates from asymptomatic, ulcer and gastritis patients by urease gene polymorphism.

The gastric-adapted bacterium Helicobacter pylori plays an important role in gastritis and ulcer disease, but no phenotypic typing scheme presently exists for this organism. With a view to the development of genotypic typing, we have compared isolates of H. pylori from gastritis or ulcer patients with those from subjects exhibiting no disease. Variation was analysed at the urease genes, ureA and ureCD, by employing PCR-generated probes in genomic Southern blot hybridizations. Whilst ureA restriction fragments provided a fourfold subgrouping of strains, ureCD fragments were considerably more discriminatory. Twenty-four combined ureACD profiles were generated with Hind III, subdividing the 64 strains into 11 types and 13 single profiles. The most prevalent profile (UI) was found in 33% of strains, almost all from gastritis or ulcer patients. On the other hand strains isolated from asymptomatic individuals had the most diverse ureACD profiles. A key finding from this set of isolates was that strains of H. pylori associated with general gastroduodenal disease were genetically more homogeneous than strains carried by people without disease symptoms.

Bacterial Typing Techniques↗
PubMed2006

The nickel site of Bacillus pasteurii UreE, a urease metallo-chaperone, as revealed by metal-binding studies and X-ray absorption spectroscopy.

UreE is a homodimeric metallo-chaperone that assists the insertion of Ni(2+) ions in the active site of urease. The crystal structures of UreE from Bacillus pasteurii and Klebsiella aerogenes have been determined, but the details of the nickel-binding site were not elucidated due to solid-state effects that caused disorder in a key portion of the protein. A complementary approach to this problem is described here. Titrations of wild-type Bacillus pasteurii UreE (BpUreE) with Ni(2+), followed by metal ion quantitative analysis using inductively coupled plasma optical emission spectrometry (ICP-OES), established the binding of 2 Ni(2+) ions to the functional dimer, with an overall dissociation constant K(D) = 35 microM. To establish the nature, the number, and the geometry of the ligands around the Ni(2+) ions in BpUreE-Ni(2), X-ray absorption spectroscopy data were collected and analyzed using an approach that combines ab initio extended X-ray absorption fine structure (EXAFS) calculations with a systematic search of several possible coordination geometries, using the Simplex algorithm. This analysis indicated the presence of Ni(2+) ions in octahedral coordination geometry and an average of two histidine residues and four O/N ligands bound to each metal ion. The fit improved significantly with the incorporation, in the model, of a Ni-O-Ni moiety, suggesting the presence of a hydroxide-bridged dinuclear cluster in the Ni-loaded BpUreE. These results were interpreted using two possible models. One model involves the presence of two identical metal sites binding Ni(2+) with negative cooperativity, with each metal ion bound to the conserved His(100) as well as to either His(145) or His(147) from each monomer, residues found largely conserved at the C-terminal. The alternative model comprises the presence of two different binding sites featuring different affinity for Ni(2+). This latter model would involve the presence of a dinuclear metallic core, with one Ni(2+) ion bound to one His(100) from each monomer, and the second Ni(2+) ion bound to a pair of either His(145) or His(147). The arguments in favor of one model as compared to the other are discussed on the basis of the available biochemical data.

Absorptiometry, Photon↗
PubMed2006

Multiple isotope effect study of the hydrolysis of formamide by urease from jack bean (Canavalia ensiformis).

Multiple kinetic isotope effects have been measured for the urease-catalyzed hydrolysis of formamide at pH 6.0 and 25 degrees C. These kinetic isotope effects include the carbonyl-C ((13)k = 1.0241 +/- 0.0009), the carbonyl-O ((18)k = 0.9960 +/- 0.0009), the formyl-H ((D)k = 0.95 +/- 0.01), the leaving-N ((15)k= 1.0327 +/- 0.0006), and the nucleophile-O ((18)k = 0.9778 +/- 0.0005). In addition, the enzyme does not catalyze the exchange of oxygen from the solvent into the carbonyl-O of formamide or the product, formate ion. The isotope effects are consistent with the rate-determining collapse of the tetrahedral intermediate (i.e., C-N bond cleavage). The pH optimum for formamide is at pH 5.3, whereas for urea, it is near 8.0. This is best accommodated by the mechanism proposed by Hausinger and Karplus, in which an active site cysteine binds to the nonleaving nitrogen in urea. For urea, the preference is for the anionic form of the sulfhydryl; for formamide, the neutral form is preferred, leading to the lower pH optimum.

Canavalia↗
PubMed2002

Hydrolytically active tetranuclear nickel complexes with structural resemblance to the active site of urease.

Reaction of the new asymmetric ligand 2-(N-isopropyl-N-((1-methylimidazolyl)methyl)aminomethyl)-6-(N-carboxylmethyl-N-((1-methylimidazolyl)methyl) aminomethyl)-4-methylphenol (ICIMP) with nickel perchlorate and diphenylacetic acid leads to the formation of tetranuclear nickel complexes, whose crystal structures reveal that they consist of dimers of dimers in which each Ni(2) unit has a coordination environment that is similar to the active site of urease. One complex has been shown to coordinate urea and catalyze the hydrolysis of an organophosphate monoester.

Bacillus↗
PubMed2004

Molecular details of urease inhibition by boric acid: insights into the catalytic mechanism.

The structure of the complex of urease, a Ni-containing metalloenzyme, with boric acid was determined at 2.10 A resolution. The complex shows the unprecedented binding mode of the competitive inhibitor to the dinuclear metal center, with the B(OH)3 molecule bridging the Ni ions and leaving in place the bridging hydroxide. Boric acid can be considered a substrate analogue of urea, and the structure supports the proposal that the Ni-bridging hydroxide acts as the nucleophile in the enzymatic process of urea hydrolysis.

Binding Sites↗
PubMed2000

Recurrence of gastric ulcer dependent upon strain differences of Helicobacter pylori in urease B gene.

To evaluate the role of different strains of Helicobacter pylori on the recurrence of gastric ulcer, we divided H. pylori into four types (I, II, III, and IV) according to the urease B gene using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The relationship between the recurrence of gastric ulcer and the prevalence of H. pylori types was studied in 32 patients with benign open gastric ulcers using upper gastrointestinal endoscopy. The rate of recurrence was significantly lower in patients with type II than in patients with types I, III, and IV (P<0.05). Using Mongolian gerbils, an animal model of H. pylori infection, we also showed that the occurrence of gastric ulceration following restraint water-immersion stress was significantly lower in type II compared with types I and III. These data indicate that in the context of ulcer recurrence, it is not necessary to eradicate H. pylori during infection with type II.

Aged↗
PubMed2000

Diagnosis of Helicobacter pylori infection from antral biopsies in pediatric patients is urease test that reliable?

Our objectives were to determine if the urease (CLO) test alone is a reliable diagnostic test for H. pylori gastritis in children and if the density of H. pylori influences the CLO test result. We performed a combined retrospective and prospective study reviewing the results of CLO-test and histology of gastric mucosal biopsy from 67 patients (35 females) with H. pylori gastritis. Two antral biopsies were inoculated on the CLO-test and two processed for histology to grade the severity of gastritis and H. pylori density. The mean age of patients was 11 years (SD +/- 4.53). Only 50 patients tested positive for H. pylori on CLO-test, whereas all patients were positive on histology. The sensitivity of the CLO-test was 75%. There was a significant association between CLO-test positivity and the density of H. pylori organisms on histology (P < 0.01), and with the severity of gastritis (P < 0.001) by the Pearson chi-square test. However, there was no association between the density of H. pylori and severity of gastritis. In conclusion, the CLO-test is not reliable as a sole diagnostic test for H. pylori gastritis in children because of a significant number of false negatives. Histologic examination of gastric mucosal biopsy is superior to the CLO-test in diagnosing H. pylori infection.

Adolescent↗
PubMed2001

Clinical significance of oral urease in diagnosis of Helicobacter pylori infection by [13C]urea breath test.

This study was performed to evaluate the effect of oral flora on [13C]urea breath test in detecting H. pylori infection and find an optimal method and timing for sample collection. Forty-five volunteers were included in this study. The [13C]urea breath test was performed using mouthwash, endoscopic administration, and conventional methods. According to the receiver-operating characteristic curves, the earliest optimal time for discriminating H. pylori-positive and H. pylori-negative patients was at 25 min with the mouthwash method with 78% sensitivity and 82% specificity, at 2 min with the endoscopic administration method with 100% sensitivity and 100% specificity, and at 6 min with the conventional method with 100% sensitivity and 95% specificity. The study shows a significant effect of oral urease on the results of the [13C]urea breath test. The timing of sampling collection can be shortened to 6 min with the conventional method or to 2 min through endoscopic administration.

Adult↗
PubMed2025

Leclercia barmai sp. nov., isolated from worm castings of Eisenia fetida, is a urease-positive, 3-nitropropionic acid and glycerol-consuming bacterium.

A comprehensive polyphasic characterization has validated the unique taxonomic position of a novel bacterium, strain EMC7T, isolated from the worm castings of earthworm, Eisenia fetida, collected from the Centre for Floriculture and Agri-Business Management (COFAM), NBU (26.7072&#xb0; N, 88.3554&#xb0; E). Whole-genome sequence of this Gram-stain-negative, facultatively anaerobic, motile, rod-shaped bacterium showed maximum sequence homology with Leclercia adecarboxylata NBRC 102595T, placing it within the genus Leclercia. The genome of EMC7T is 5.03 Mbp with a G&#x2009;+&#x2009;C content of 56.3&#xa0;mol%. Phylogenetic analyses established its distinctiveness from Leclercia adecarboxylata and Leclercia tamurae. DNA-DNA hybridization (dDDH) value was 23.6%, and the average nucleotide identity (ANI) was 82.1%, both below the thresholds for prokaryotic species differentiation. Predominant fatty acids were C16:0 (29.53%), summed feature 3 (C16:1&#x3c9;7c/C16:1&#x3c9;6c, 16.51%), and C18:1&#x3c9;7c (10.90%). Notably, EMC7T exhibited urease activity and could metabolize 3-nitropropionic acid (3-NPA), glycerol, tellurite, selenate, and selenite, suggesting potential bioremediation applications. Biochemical tests, phenotypic traits, genotypic data, and physiological properties cumulatively differentiated EMC7T from its closest relatives. Based on chemotaxonomic, phenotypic, genomic, and phylogenetic evidence, strain EMC7T represents a novel bacterial species of the genus Leclercia, for which the name Leclercia barmai sp. nov. (type strain EMC7T&#x2009;=&#x2009;MCC 5183T&#x2009;=&#x2009;JCM 36544T) is proposed.

Animals↗
PubMed1991

Determination of urea in serum by using naturally immobilized urease in a flow injection conductimetric system.

A flow injection method was developed, aimed at the determination of urea in human serum. The system makes use of the naturally immobilized urease present in Canavalia ensiformis DC (jack bean). A column is filled with small pieces of this bean, and the sample (50 microliters) containing urea passes through it carried by a 1% NaCl solution. On leaving the column the stream is merged with an alkaline reagent (0.5 mol dm-3 NaOH; 0.5% disodium dihydrogen ethylenediaminetetraacetate). The ammonium ions, arising from the enzymatic reaction that occurs inside the column, are changed into the molecular form, which permeates a polytetrafluoroethylene membrane and is received in a de-ionized water acceptor stream. The ammonia ionizes causing an increase in the conductance, which is proportional to the urea content of the sample. About 40 samples can be processed in 1 h with negligible carry-over and with a relative standard deviation of 1% or less. The results are in agreement with those obtained by a standard spectrophotometric method.

Conductometry↗
PubMed2005

Precipitation of Zn2Al LDH by urease enzyme.

A biomineralization process based on the promotion of precipitating agent by the urea-urease enzymatic system is developed to prepare ZnAl layered double hydroxide materials and the effects of the enzymatic reaction parameters on the structural and textural properties of the materials are investigated on the basis of XRD and EM analysis.

Aluminum Compounds↗
PubMed1971

Radiation-target molecular weights of urease and of L-glutamate dehydrogenase, and their relevance to the size of the functional subunits.

Activities of the oligomeric enzymes urease and l-glutamate dehydrogenase were measured after exposure as dry preparations to various doses of electron radiation. Inactivation curves were exponential. ;Target sizes' deduced from these were small compared with the molecular weights of the whole enzyme molecules, but accorded well with independent estimates of the sizes of functional subunits. It was concluded that, when these were in associated from, there could be no transfer of absorbed energy between subunits.

Air↗
PubMed1984

The nickel ion environment in jack bean urease.

Preliminary results of an extended X-ray absorption fine structure (e.x.a.f.s.) and X-ray absorption near edge structure study of jack bean urease have recently been reported [Hasnain & Piggott (1983) Biochem. Biophys. Res. Commun. 112, 279]. These results indicate that the environment of the nickel ion in the enzyme is similar to that in the model compounds Ni(L)2(L')1(ClO4)1 (where L is 1-n-propyl-2-alpha-hydroxybenzylbenzimidazole and L' is the deprotonated form) and Ni(HMB)3(Br)2 (where HMB is 2-hydroxymethylbenzimidazole), the closest similarity being with Ni(L)2-(L')1(ClO4)1. A detailed e.x.a.f.s. analysis has now been carried out and the crystal structures of the two model compounds solved. These results are reported here.

Fabaceae↗
PubMed1999

Urease-positive thermophilic strains of Campylobacter isolated from seagulls (Larus spp.).

Three strains of urease-positive thermophilic Campylobacter (UPTC), designated A1, A2 and A3, were identified by biochemical characterization after isolation from faeces of seagulls in Northern Ireland in 1996. The biochemical characteristics of the strains were identical to those of strains described previously. Analysis by pulsed-field gel electrophoresis (PFGE) after separate digestion with ApaI and SmaI demonstrated that the respective PFGE profiles were indistinguishable. The PFGE analysis also suggested that the genomes were approximately 1810 kb in length. This is the first example of the isolation of UPTC from flying homoiothermal animals, i.e. from seagulls (Larus spp.).

Animals↗
PubMed2001

The effects of UVB and temperature on the survival of natural populations and pure cultures of Campylobacter jejuni, Camp. coli, Camp. lari and urease-positive thermophilic campylobacters (UPTC) in surface waters.

AIMS: To determine whether diurnal and seasonal variations in campylobacters in surface waters result from the effects of temperature and u.v. radiation, and whether natural populations of Campylobacter lari and urease-positive thermophilic campylobacters (UPTC) from birds survive better in surface waters than Camp. jejuni from sewage. METHODS AND RESULTS: Natural populations of Camp. lari and UPTC in sea water, and Camp. jejuni in river water, were exposed to artificial sunlight (equivalent to a sunny day in June). Both populations became non-culturable within 30 min, with T90s of 15 min and 25 min, respectively. Cultures of Camp. jejuni became non-culturable within 40 min and those of Camp. coli, Camp. lari and UPTC, within 60 min. In darkness, survival was temperature-dependent. Natural populations took 12 h at 37 degrees C and 5 days at 4 degrees C to become non-culturable in sea water, and slightly less in river water. Cultures of Camp. lari and UPTCs survived for significantly longer than Camp. jejuni and Camp. coli. Loss of culturability for all isolates was most rapid at 37 degrees C and slowest at 4 degrees C. Newly isolated strains from sea water and river water behaved in an almost identical manner to NCTC strains. CONCLUSION: Campylobacter lari and UPTCs survive for longer in surface waters than Camp. jejuni and Camp. coli, particularly in the dark. Low Campylobacter numbers in coastal waters in the summer, especially in the afternoon, are due to the combined effects of higher temperatures and higher levels of u.v. radiation. SIGNIFICANCE AND IMPACT OF THE STUDY: Campylobacter lari and UPTCs from birds predominate in bathing waters in Morecambe Bay because they are better able to survive; they also originate from closer to the shore than Camp. jejuni and Camp. coli in sewage effluent, which survive poorly and die before the incoming tide reaches the shore. The predominance of Camp. jejuni in river water results from its dominance of the inputs and not from its ability to survive.

Animals↗