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PubMed2001

Structural basis for Ni(2+) transport and assembly of the urease active site by the metallochaperone UreE from Bacillus pasteurii.

Bacillus pasteurii UreE (BpUreE) is a putative chaperone assisting the insertion of Ni(2+) ions in the active site of urease. The x-ray structure of the protein has been determined for two crystal forms, at 1.7 and 1.85 A resolution, using SIRAS phases derived from a Hg(2+)-derivative. BpUreE is composed of distinct N- and C-terminal domains, connected by a short flexible linker. The structure reveals the topology of an elongated homodimer, formed by interaction of the two C-terminal domains through hydrophobic interactions. A single Zn(2+) ion bound to four conserved His-100 residues, one from each monomer, connects two dimers resulting in a tetrameric BpUreE known to be formed in concentrated solutions. The Zn(2+) ion can be replaced by Ni(2+) as shown by anomalous difference maps obtained on a crystal of BpUreE soaked in a solution containing NiCl(2). A large hydrophobic patch surrounding the metal ion site is surface-exposed in the biologically relevant dimer. The BpUreE structure represents the first for this class of proteins and suggests a possible role for UreE in the urease nickel-center assembly.

Bacillus↗
PubMed1994

Relationship of decreased caecal urease activity by dietary penicillin to nitrogen utilisation in chickens fed on a low protein diet plus urea.

1. The relationship of the decreased caecal urease activity by dietary penicillin to nitrogen utilisation was assessed in chickens fed a low protein diet plus urea. 2. Dietary penicillin at 20 and 100 mg/kg decreased anaerobic bacteria counts, urease activity and ammonia concentration in caecal contents (P < 0.05, except for ammonia in the case of the 100 mg/kg penicillin diet). 3. The 20 mg/kg penicillin diets significantly increased the excretion of urea and total nitrogen (P < 0.05) and decreased ammonia excretion, and significantly reduced nitrogen retention (P < 0.05). The 100 mg/kg penicillin diet also resulted in similar but not significant changes, which tended to be less than those by the 20 mg/kg penicillin diet. 4. Ammonia, urea, glutamine and uric acid concentrations in blood, liver and kidney were unchanged by dietary penicillin. 5. It is concluded that caecal ammonia production from urea was closely correlated with nitrogen utilisation in chickens fed a low protein diet plus urea.

Ammonia↗
PubMed2006

Boric acid and boronic acids inhibition of pigeonpea urease.

Urease from the seeds of pigeonpea was competitively inhibited by boric acid, butylboronic acid, phenylboronic acid, and 4-bromophenylboronic acid; 4-bromophenylboronic acid being the strongest inhibitor, followed by boric acid > butylboronic acid > phenylboronic acid, respectively. Urease inhibition by boric acid is maximal at acidic pH (5.0) and minimal at alkaline pH (10.0), i.e., the trigonal planar B(OH)3 form is a more effective inhibitor than the tetrahedral B(OH)4 -anionic form. Similarly, the anionic form of phenylboronic acid was least inhibiting in nature.

Boric Acids↗
PubMed1980

[Studies on the effect of phosphoric phenyl ester diamide as inhibitor of the rumen urease of dairy cows. 1. Influence on urea hydrolysis, ammonia release and fermentation in the rumen].

An amount of 0, 0.1, 0.5 and 1.0% (related to N) of phosphoric phenyl ester diamide (PPD) effective as urease blocking substance was applied to the surface of urea with oilbitumen and fed to cows with rumen cannulae over a period of 31 resp. 164 ... 167 days. (The ration essentially consisted of 4.5 kg dried roughage and 1.5 kg starch or 1.1 kg starch plus 0.4 kg sugar and contained 50 g urea). By treating the urea with PPD, the activity of urease, the hydrolysis rate of urea and the NH3-concentration in the rumen were significantly diminished 0.5 to 2 hours after feeding (alpha = 0.05). The effect of PPD was greatest in the first days and decreased with the advancing feeding period. In the variant with 0.5% PPD the examined parameters were significantly reduced after 142 to 164 days, too. This effect remained traceable in its diminished form even after the preparation was discontinued over a period of 35 days. The dynamics of the NH3-concentration was not altered by PPD after a longer feeding period. One can conclude that PPD inhibits the hydrolysis of urea but does not retard it. In conclusion one can say that, because of PPD, the toxicity of urea is lower without the utilisation of urea being better. Due to PPD the molar propionate level in volatile fatty acids decreases significantly and the acetate-propionate relation is expanded.

Ammonia↗
PubMed1980

[Studies on the effect of phosphoric phenyl ester diamide as inhibitor of the rumen urease of dairy cows. 2. The metabolism of 15N-urea].

Two lactating dairy cows supplied with rumen and duodenal re-entrance cannulae received with their rations containing 8.2% vegetable crude protein 180 g urea per day (I) or urea treated with the urease inhibitor phosphoric phenyl ester diamide (PPD, 1% of the N-quota). The cows, accustomed to urea, received the PPD-urea without adaptation (II) and after a 30-day adaptation (III) to PPD. On the first day of the experiment one half of the urea was given ruminally in its 15N-labelled form. 2 h after the isotope supplementation 62.5 and 24 mg NH3 and 6.58 and 21 mg urea/100 ml could be detected in the rumen juice of I to III. Within 72 h 16.6, 26.1 and 25.2% of the 15N-excess given (15N') passed the duodenum in TCA - soluble form and 31.2, 28.4 and 41.7% in TCA - precipitable form. 15.6, 24.4 and 21.5% of the total amount of 15N were excreted in urine and 4.5, 4.6 and 6.0% in the milk protein. The values for faeces were 14.4, 14,4 and 15.4%. The conclusion from these results and from the dynamics of the relative 15N' in the fractions of the rumen fluid is that with a limited inhibition of rumen urease by PPD as it develops after the adaptation, the utilisation of urea-N can be improved.

Animal Feed↗
PubMed1997

Protein degradability, amino acid composition, trypsin inhibitor and urease activity of raw and heat-treated fullfat soybean.

The effects of different heat treatments were studied on chemical composition, protein degradability, amino acid composition, trypsin inhibition and urease activity. Three lactating Holstein cows fitted with rumen cannulae were used. Fullfat soybean was prepared employing different forms of heat treatment: dry-extrusion at 150 degrees C for 25 s (treatment I); wet-extrusion at 95 degrees C for 30 min (treatment II); toasted soybean at 105 degrees C for 30 min (treatment III) extracted soybean meal (treatment IV); and untreated soybean (treatment V). The incubation times were 0, 2, 4, 8, 16, 24 and 48 h. Samples of raw and heat-treated soybean before incubation and the undegraded fraction after 4 and 16 h of incubation were analyzed for amino acids. The results showed that heat treatments did not modify chemical composition, but significantly reduced the content of trypsin inhibition and urease activity, as well as decreased protein degradability. The dry extrusion technique was comparatively the most effective. Amino acid content was not significantly influenced by different techniques, but the quantity of amino acids escaping degradation in the rumen increased.

Amino Acids↗
PubMed1983

Photo-oxidation of Jack bean urease in the presence of methylene blue.

Photo-oxidation of Jack bean urease was performed in the presence of a low concentration of methylene blue, which led to the complete loss of the enzymatic activity. The inactivation was more remarkable in an alkaline region than in an acidic region and prevented by the addition of histidine or methionine. Amino acid analysis of the oxidized enzyme revealed that the number of histidine residues had decreased to 73% that of the native enzyme, but the numbers of other amino acid residues were not significantly affected. Benzohydroxamic acid, a specific urease inhibitor, protected the active site of the enzyme against photo-oxidation. On the other hand, oxidation of the enzyme decreased its binding ability with caprylo- and benzohydroxamic acid to one-third. These results suggest that histidine residues are modified by photo-oxidation and are essential to both the enzymatic activity and the binding ability with hydroxamic acid.

Fabaceae↗
PubMed1984

Influence of monensin and lasalocid on mineral metabolism and ruminal urease activity in steers.

Growing steers were used in a replicated 3 X 3 Latin square to study the influence of ionophores on mineral metabolism and ruminal urease activity. Treatments consisted of: 1) basal high energy diet; 2) basal plus 33 ppm lasalocid and 3) basal plus 33 ppm monensin. Each period was 33 days and apparent absorption and retention of macrominerals were measured during the last 5 days of each period. Mineral intake during the collection period was not affected by treatment. Both ionophores increased apparent absorption of sodium, magnesium and phosphorus. Retention of magnesium and phosphorus were higher for steers receiving either lasalocid or monensin. Potassium and calcium absorption were not significantly affected by treatment. Serum concentrations of macrominerals were similar for all treatments. Zinc and copper concentrations in serum were higher in animals fed monensin or lasalocid. Steers fed either ionophore had lower concentrations of soluble potassium and calcium in rumen fluid. Both ionophores also decreased ruminal osmolality. Bacterial urease, a nickel-dependent enzyme, was decreased by 28 and 66% in animals that received lasalocid and monensin, respectively. These findings indicate that lasalocid and monensin affect metabolism of certain minerals in ruminants.

Animals↗
PubMed2007

Comparison of the 13C-urea blood test to histology and rapid urease testing in the diagnosis of Helicobacter pylori infection in children.

OBJECTIVE: Helicobacter pylori infection is commonly investigated in children with abdominal pain. The definitive means of diagnosing infection, histology, requires endoscopy and sedation, making it invasive and expensive. Our objective was to compare histology against a less invasive and safer method, the 13C-urea blood test. PATIENTS AND METHODS: Forty children with abdominal pain undergoing upper endoscopy were randomized into either of 2 dosages of 13C-urea. Several biopsies were taken for histology and rapid urease testing. After endoscopy, each child ingested a randomly assigned dosage of either 75 mg or 125 mg 13C-urea, and blood was withdrawn 30 min later. RESULTS: Irrespective of the dosage of 13C-urea, the 13C-urea blood test performed with high accuracy (89%) when compared against either histology or rapid urease testing. The sensitivity and specificity of the blood test was 83% and 91%, respectively. When the smaller dosage of 13C-urea was used, the accuracy of the blood test was 100% compared with histology. There were no adverse events related to using either dosage of 13C-urea. CONCLUSIONS: The 13C-urea blood test may be comparable with histology in diagnosing H. pylori infection in children, and the smaller dosage of 13C-urea does not adversely affect blood test performance. The 13C-urea blood test is well tolerated in children.

Adolescent↗
PubMed1998

Emended description of Campylobacter sputorum and revision of its infrasubspecific (biovar) divisions, including C. sputorum biovar paraureolyticus, a urease-producing variant from cattle and humans.

A polyphasic taxonomic study of 15 bovine and human strains assigned to the catalase-negative, urease-positive campylobacter (CNUPC) group identified these bacteria as a novel, ureolytic biovar of Campylobacter sputorum for which we propose the name C. sputorum bv. paraureolyticus: suitable reference strains are LMG 11764 (human isolate) and LMG 17590 (= CCUG 37579, bovine isolate). The present study confirmed previous findings showing that the salient biochemical tests used to differentiate C. sputorum bv. sputorum from C. sputorum bv. bubulus are not reproducible; and that the absolute validity of source-specific biovars of the species is questionable. A correlation between the results of numerical analysis of protein profiles and the reaction of strains in certain enzyme tests was, however, noted. Therefore, it is proposed that the infrasubspecific (biovar) divisions of C. sputorum should be revised to include bv. sputorum for catalase-negative strains; bv. fecalis for catalase-positive strains; and bv. paraureolyticus for urease-positive strains. Strains classified previously as bv. bubulus should be reclassified as bv. sputorum. The species description of C. sputorum is revised accordingly.

Animals↗
PubMed2002

Susceptibility of Helicobacter pylori and its urease activity to the peroxidase-hydrogen peroxide-thiocyanate antimicrobial system.

The susceptibility of Helicobacter pylori to the antimicrobial system involving lactoperoxidase, hydrogen peroxide and thiocyanate was investigated. The inhibitory effect of the system on the urease activity of H. pylori, which plays a role in its colonisation of the stomach, was also investigated. Twelve H. pylori strains examined, including 10 clinical isolates, were all inhibited by the peroxidase system in brain-heart infusion broth supplemented with fetal calf serum, but to different extents. The killing effect was observed within 3 h. Although bacterial viability recovered afterwards, there was still a clear difference between cultures incubated in the presence of the complete system and control cultures incubated in the absence of lactoperoxidase, after incubation for 24 h. The urease activity and viability of H. pylori were both inactivated by this system in phosphate buffer. These effects were dependent on the concentrations of both lactoperoxidase and hydrogen peroxide and were abolished by the addition of cysteine. Furthermore, these effects were observed when bovine lactoperoxidase was replaced by recombinant human lactoperoxidase or native or recombinant human myeloperoxidase. The peroxidase system found in saliva and milk may contribute to the host defence against H. pylori infection and inhibition of transmission via the oral route.

Animals↗
PubMed1987

Preliminary characterization of the urease and a 96 kDa surface-expressed polypeptide of Ureaplasma urealyticum.

Analysis by SDS-PAGE of Ureaplasma urealyticum (predominantly serotype 8), propagated in a growth medium containing 10% (v/v) foetal calf serum, revealed a complex series of polypeptides apparently free of medium contaminants. Serological analysis using an immobilized antibody reagent, and immunoblotting using a polyclonal serum, showed the presence of two major and several minor antigens. One major antigen, a putative surface component of apparent molecular mass 96 kDa was shown, with a monoclonal antibody, to be serotype-specific. Growth of the organism was partially suppressed in the presence of the antibody. The second major antigen had an apparent molecular mass of 76 kDa and was presumed to be an internal component since it failed to label with the Bolton and Hunter reagent, in contrast to the 96 kDa antigen. Another monoclonal antibody was characterized which detected the canonical urease enzyme of the organism serotype 8 and of the two other human serotypes tested. Purification of this urease antigen by affinity chromatography and electrophoretic analysis of polypeptides after denaturation revealed a single polypeptide of molecular mass 76 kDa, putatively related to the above major antigen. Enzymic activity could be recovered after purification and demonstrated by in situ techniques only when electrophoretic analysis was done under non-denaturing conditions suggesting that the functional enzyme is a multimeric complex.

Antigens, Bacterial↗
PubMed1993

Evaluation of the polymerase chain reaction for detecting the urease C gene of Helicobacter pylori in gastric biopsy samples and dental plaque.

A polymerase chain reaction (PCR) assay with oligonucleotide primers homologous to a portion of the urease C gene of Helicobacter pylori was evaluated for specificity with pure DNA and biopsy material. The assay was used to test for the presence of the organism in dental plaque. The species specificity of detection was confirmed by ensuring that the primers did not amplify DNA extracts from H. cinaedi, H. felis, H. fennelliae, H. mustelae and H. nemestrinae. Sixty-two gastric biopsy samples collected from 14 patients (antrum, body and duodenal sites) were cultured and PCR was performed on the samples after culture. Primer sites were conserved in genomically diverse strains. Samples prepared by single-step heat lysis of bacterial cells and biopsy material did not inhibit PCR. The overall specificity was 96% irrespective of genotype. H. pylori was not cultured from dental plaque (15 patients), neither was H. pylori DNA detected by PCR in either urea breath test-positive or -negative individuals. The results showed that primer pair sequences within the urease C gene are conserved in most strains and provide an accurate basis for detecting H. pylori. As the PCR assay was not inhibited and did not yield false positive results with crude extracts from organisms or in the presence of biopsy material, its value as a diagnostic test was confirmed.

Base Sequence↗
PubMed1996

Factors affecting growth and antibiotic susceptibility of Helicobacter pylori: effect of pH and urea on the survival of a wild-type strain and a urease-deficient mutant.

This study investigated how pH and the presence of urea affect the survival and growth of Helicobacter pylori and whether these factors affect susceptibility to antibiotics in vitro. The viability of a wild-type strain and a urease-deficient mutant of H. pylori was studied after incubation for 1 h in buffers at different pH values at 37 degrees C under microaerophilic conditions. Viable counts were not affected at pH 5 and pH 7. In buffer at pH 3, there were no viable organisms, but urea (6.25 mM) protected the wild-type strain, which survived well. At pH 9, urea further reduced the viability of H. pylori and flurofamide almost abolished the effect of urea on the wild-type strain. Neither urea nor flurofamide affected the viability of the urease-deficient mutant under the same conditions. Growth was also pH dependent and was enhanced in shake-cultures. At pH 5, urea supported growth of the wild-type strain, but at pH 7 a toxic effect on the bacteria was observed. Growth of H. pylori at pH 5.9 was poor, and susceptibility to amoxycillin, erythromycin and clarithromycin was markedly less than at pH 7.2 and 7.9. The bactericidal activities of metronidazole and tetracycline were similar at the different pH values studied. At neutral pH the killing rates of amoxycillin and clarithromycin were growth rate dependent. Susceptibility to metronidazole was enhanced in stationary cultures. The interaction obtained between the proton pump inhibitor, omeprazole, and amoxycillin at pH 7 was of additive type. These results suggest that pH and growth conditions may be important in the antibacterial efficacy of different antibiotics in vivo and also provide a possible explanation for the potentiating effect of omeprazole with antibiotics in the treatment of H. pylori infections.

Amoxicillin↗
PubMed1996

Biovar-specific epitopes of the urease enzyme of Ureaplasma urealyticum.

The importance of Ureaplasma urealyticum as a pathogen in premature neonates and patients with a profound defect in humoral immunity has, over the last few years, become well recognised. U. urealyticum is unique amongst the Mycoplasmataceae for its use of urea metabolism as an essential source of energy. The urease enzyme responsible for this is, therefore, of prime importance and any variability in expression of this enzyme may play a role in virulence of the organism. U. urealyticum is divided into 14 serovars comprising two biovars -- the parvo-biovar and T960-biovar. In this study monoclonal antibodies (MAbs) were produced against the urease enzyme. Two distinct epitopes of the 72-kDa alpha-subunit were recognised by three different MAbs. Under denaturing conditions both epitopes were shown to be specific for the parvo-biovar.

Amino Acid Sequence↗
PubMed2004

Description of 'Candidatus Helicobacter heilmannii' based on DNA sequence analysis of 16S rRNA and urease genes.

While Helicobacter pylori is accepted as the major bacterial agent of gastric disease in humans, some patients and many animals are infected with a larger, tightly helical-shaped bacterium previously referred to as 'Helicobacter heilmannii' or 'Gastrospirillum hominis'. Taxonomic classification of these bacteria has been hampered by the inability to cultivate them in vitro and by the inadequate discriminatory power of 16S rRNA gene sequence analysis. This study describes the detection and phylogenetic analysis of 26 different gastrospirillum isolates from humans and animals, which incorporates sequence data based on the 16S rRNA and urease genes. Fifteen gastrospirilla detected in humans, primates and pigs clustered with 'Candidatus Helicobacter suis', thus expanding the host range for this organism. By comparison, based on 16S rRNA data, the remaining 11 gastrospirilla could not be differentiated from Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis. However, urease gene sequence analysis allowed for the discrimination of this latter group into four discrete clusters, three of which contained the above recognized species. The fourth cluster contained isolates from human and feline hosts, and should provisionally be considered a unique bacterial species, for which the name 'Candidatus Helicobacter heilmannii' is proposed.

Adult↗
PubMed2004

Effect of dietary anti-Helicobacter pylori-urease immunoglobulin Y on Helicobacter pylori infection.

Recently, chicken egg yolk was recognized as an inexpensive antibody source, and the therapeutic usefulness of egg yolk immunoglobulin Y (IgY) in oral passive immunization has been investigated. Although multiple antibiotic treatments eradicate most Helicobacter pylori (H. pylori) infections, therapy fails in 10-15% of cases due to the development of drug resistance. Consequently, it is important that new, more broadly based therapies for the treatment of H. pylori infection should be identified. The present study evaluated the effect, on H. pylori infection, of IgY prepared from egg yolk of hens immunized with H. pylori urease (anti-HpU IgY). Seventeen asymptomatic volunteers diagnosed as H. pylori-positive by the 13C-urea breath test (UBT) were orally administered anti-HpU IgY for 4 weeks. Four weeks later, UBT values were significantly decreased although no case showed H. pylori eradication. An H. pylori-positive 53-year-old female gastritis patient administered anti-HpU IgY plus lansoprazole for 8 weeks showed a decrease in serum pepsinogen (PG) I and UBT values as well as an increase in the PG I/II ratio. In conclusion, anti-HpU IgY may mitigate H. pylori-associated gastritis and partially attenuate gastric urease activity. Furthermore, anti-HpU IgY combined with antacids appears to ameliorate gastric inflammation. These encouraging results may represent a novel approach to the management of H. pylori-associated gastroduodenal disease.

Adult↗