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Effect of hydroxamic acids on growth and urease activity in Corynebacterium renale.

Studies were conducted on the effect of four different hydroxamic acids (HA), hydroxyurea, acetohydroxamic acid, p-flurobenzoylhydroxamic acid and sorbylhydroxamic acid, on the growth and urease activity of Corynebacterium renale. The addition of each of these HA, at concentrations ranging form 10(-3) to 10(-5) M, to medium containing urea as the sole nitrogen source resulted in a lengthened lag period of growth the extent of which depended upon the concentration of each HA tested as well as the structure of the compound; that is, the size and (or) complexity of the side chain attached to the common terminal group of the molecule. However, the maximal growth levels achieved following conclusion of the exponential phase were not affected by the HA. Investigations on the effect of these HA on the urease activity of intact cells as well as cell-free extracts revealed that in each case the enzymatic activity was inhibited by each of the HA tested. The extent of inhibition with the intact cells was aobut one-half of that observed with cell-free extracts. Direct incubation of cell-free extracts as well as intact cells with each of the HA tested was required for maximal inhibition.

Cell-Free System↗

Biochemo-mechanical function of urease-loaded gels.

A gel system is developed that undergoes a reversible volume phase transition in response to a small amount of urea. An N-isopropylacrylamide gel in which urease is immobilized by entrapping changes its equilibrium volume discontinuously when urea molecules are hydrolyzed by urease causing a change in pH that alters the osmotic balance of the gel triggering the phase transition. The system demonstrates a method of mechano-biochemical transformation where molecular recognition and biochemical reaction are achieved by an enzyme and the macroscopic amplification of the reaction is carried out by a gel capable of a volume phase transition. The work presented here is dedicated to Professor Allan S. Hoffman to honor his 60th birthday and his pioneering contribution to the science and technology of polymer gels, both as a scientist and as an educator.

Acrylamides↗

Local pH elevation mediated by the intrabacterial urease of Helicobacter pylori cocultured with gastric cells.

Helicobacter pylori resists gastric acidity by modulating the proton-gated urea channel UreI, allowing for pH(out)-dependent regulation of urea access to intrabacterial urease. We employed pH- and Ca(2+)-sensitive fluorescent dyes and confocal microscopy to determine the location, rate, and magnitude of pH changes in an H. pylori-AGS cell coculture model, comparing wild-type bacteria with nonpolar ureI-deletion strains (ureI-ve). Addition of urea at pH 5.5 to the coculture resulted first in elevation of bacterial periplasmic pH, followed by an increase of medium pH and then pH in AGS cells. No change in periplasmic pH occurred in ureI-deletion mutants, which also induced a slower increase in the pH of the medium. Pretreatment of the mutant bacteria with the detergent C(12)E(8) before adding urea resulted in rapid elevation of bacterial cytoplasmic pH and medium pH. UreI-dependent NH(3) generation by intrabacterial urease buffers the bacterial periplasm, enabling acid resistance at the low urea concentrations found in gastric juice. Perfusion of AGS cells with urea-containing medium from coculture at pH 5.5 did not elevate pH(in) or [Ca(2+)](in), unless the conditioned medium was first neutralized to elevate the NH(3)/NH(4)(+) ratio. Therefore, cellular effects of intrabacterial ammonia generation under acidic conditions are indirect and not through a type IV secretory complex. The pH(in) and [Ca(2+)](in) elevation that causes the NH(3)/NH(4)(+) ratio to increase after neutralization of infected gastric juice may contribute to the gastritis seen with H. pylori infection.

Ammonia↗

Failure of L-carnitine to protect mice against hyperammonemia induced by ammonium acetate or urease injection.

Reports indicate that L-carnitine administration before 100% lethal dose of ammonium acetate suppresses the symptoms of ammonia toxicity and prevents death in mice. However, we have been unable to confirm this observation. The cause of discrepancy between our results and the results of others was investigated with two models of hyperammonemia in mice: 1) that induced by intraperitoneal injection of urease and 2) that induced by intraperitoneal injection of ammonium acetate. L-Carnitine administration failed to protect mice against ammonia toxicity induced by intraperitoneal injection of urease. Mortality in mice treated with L-carnitine 30 min before injection of ammonium acetate was similar to that of controls pretreated with saline. Ammonia and urea levels in plasma, liver, and brain were also similar in both groups. However, the values were significantly lower than those in mice denied either pretreatment before the ammonium acetate challenge. These results indicate that pretreatment acts to reduce blood and tissue ammonia simply by diminishing the rate of absorption of the challenge, owing to the dilution of ammonium acetate upon mixing with the contents of the peritoneal cavity. Thus, any protocol that does not compare results of a putative protective agent with those obtained with an equal volume of solvents or saline runs the risk of ascribing protective property to the agent when the protection may, in fact, have been afforded by the solvent.

Absorption↗

Therapy for urolithiasis by hydroxamic acids. II. Urease inhibitory potency and urinary excretion rate of hippurohydroxamic acid derivatives.

The apparent I50 values of various hippurohydroxamic acids against urease activity of sword bean were mostly 0.5 to 2.0 microM regardless of hydrophobicity of their substituents. However, the marked increase of hydrophilicity caused by substitution of trimethoxy groups conspicuously decreased the inhibitory potency. Methylation at alpha-position of the hydroxamic acid group in these compounds remarkably decreased the inhibitory potency, probably owing to steric hindrance by the alpha-methyl group. Thenoyl-, furoyl- and nicotino-glycinohydroxamic acids which are bioisostereomers of hippurohydroxamic acid had I50 values of 0.64, 1.3 and 5.3 microM, respectively. Furthermore, the inhibitory potency of some substituted hippurohydroxamic acids against the ureolytic activity of intact Proteus mirabilis isolated from patients with urinary tract infection, were half to one-tenth of those against urease activity of sword bean. On the other hand, m- and p-nitro-, m- and p-methoxy-, m- and p-acetylamino-hippurohydroxamic acid and furoylglycinohydroxamic acid showed high urinary excretion rates of 14 to 16% of the doses administered orally to rats, while most of the others had excretion rates of about 3 to 5%.

Fabaceae↗

N-substituted hydroxyureas as urease inhibitors.

In order to seek a urease inhibitor more potent than hydroxyurea (1), its alkyl- or phenyl-substituted derivatives were synthesized and evaluated for their effect on the jack bean urease. Of 16 compounds tested, m-methyl- (10) and m-methoxy-phenyl substituted hydroxyurea (13) showed the most potent inhibitory activities against the enzyme.

Chemical Phenomena↗

Coimmobilization of urease and glutamate dehydrogenase in electrochemically prepared polypyrrole-polyvinyl sulfonate films.

Immobilization of urease and glutamate dehydrogenase enzymes in electrochemically prepared polypyrrole-polyvinyl sulfonate films (PPY-PVS) was carried out using physical adsorption and electrochemical entrapment techniques. Detailed studies on optimum pH, Fourier transform infrared spectroscopy, cyclic voltammetry, and scanning electron microscopy of the enzymes in the immobilized state were conducted. The value of the apparent Michaelis-Menten constant was experimentally determined to be 2.5 and 2.7 for physically adsorbed and electrochemically entrapped urease in PPY-PVS films, respectively.

Biosensing Techniques↗

L-usnate-urease interactions: binding sites for the ligand.

L-usnic acid inactivates urease by formation of high molecular weight aggregates which can reached by a maximum of 880 000. L-cysteine partially reverses the inactivation of stimulating the appearance of active high molecular weight polymers. The existence of two class of binding points for L-usnic acid on the urease molecule is proposed, the first showing high affinity for the ligand, related with the loss of activity, and the second, of low affinity, related to polymerization process.

Benzofurans↗

A dip-and-read test strip for the determination of mercury(II) ion in aqueous samples based on urease activity inhibition.

A sensitive dip-and-read test strip for the determination of mercury in aqueous samples based on the inhibition of urease reaction by the ion has been developed. The strip has a circular sensing zone that containing two layers: the top layer is a cellulose acetate membrane where urease is immobilized on it; the bottom layer is a pH indicator wafer that is impregnated with urea. The principle of the measurement is based on the disappearance of a yellow spot on the pH indicator wafer. The elapsing time until the disappearance of the spot which depends on the concentration of mercury(II) ion is measured with a stopwatch. Under the experimental conditions, as low as 0.2 ng/ml mercury can be observed with the detection range from 0.2 to 200 ng/ml in water. Organomercury compounds give essentially the same response as inorganic mercury. Heavy-metal ions such as Ag(I), Cu(II), Cd(II), Ni(II), Zn(II), and Pb(II) as well as other sample matrixes basically do not interfere with the mercury measurement.

Biosensing Techniques↗

Conservation of nitrogen in cattle feedlot waste with urease inhibitors.

Feedlot cattle normally retain less than 20% of their dietary nitrogen intake. Sixty to 80% of the nitrogen excreted is normally lost through volatilization of ammonia, which is primarily generated from urea. This loss of ammonia nitrogen pollutes the environment and creates an unfavorable ratio of nitrogen to phosphorous (N:P) in the waste for crop growth. Two urease inhibitors, cyclohexylphosphoric triamide (CHPT) and N-(n-butyl) thiophosphoric triamide (NBPT) were evaluated for their ability to reduce the rate of urea hydrolysis in beef cattle feedlot pens. Initially, a total of six pens were used, two pens per treatment, with approximately 70 cattle per pen, and a single topical application of CHPT or NBPT at 20 mg/kg of manure. Essentially no urea was found in untreated pens. However, with CHPT treatment, 2 g of urea/kg of dry manure accumulated by d 4, and all gradually disappeared by d 11; NBPT conserved 3 and 3.5 g of urea/kg by d 4 and 9, respectively, and it had disappeared by d 14 (treatment [trt] x day, P = .003). A second study involved application of NBPT weekly for 6 wk. This caused urea to accumulate to a peak concentration of 17 g/kg of manure by d 30 (trt x day2, P = .001). Once the treatment was stopped the urea concentration began to decrease. When the NBPT was applied weekly, the concentration of ammonia in the waste was less for the treated pens (trt x day, P = .01), the total nitrogen was greater (trt x day, P = .04), pH tended to be lower (trt x day, P = .10), and the total volatile acids were not different (trt x day, P = .51) from untreated pens. We concluded that urease inhibitors could be used to control ammonia emissions from animal wastes, prevent environmental damage, and produce a more balanced (N:P) fertilizer from manure.

Animals↗

Influence of the novel urease inhibitor N-(n-butyl) thiophosphoric triamide on ruminant nitrogen metabolism: I. In vitro urea kinetics and substrate digestion.

Two in vitro digestion experiments were conducted to evaluate the influence of the novel urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) on in vitro urea kinetics, substrate digestion, and fermentation characteristics. In Exp. 1, in vitro incubations were conducted in 50-mL test tubes containing .25 g of ground fescue hay to which 0, 6.5, 13, 26, or 52 mg of NBPT in a buffered ruminal fluid innoculum was added. Tubes were incubated in triplicate at 39 degrees C and replicated on consecutive days, with NH3 N and urea concentrations measured at 0, 10, 30, 60, 120, 240, and 360 min. Samples for VFA analysis were collected at 6 h, and incubations were continued through 48 h to estimate true digestibility (based on NDF analysis). Increasing the dose of NBPT tended (P < .12) to linearly depress the rate of urea hydrolysis and decreased (P < .0004) subsequent NH3 N formation. Although total VFA concentration at 6 h increased linearly (P < .03), acetate:propionate and estimated true digestibility decreased (P < .01) with increasing NBPT concentration. In Exp. 2, we compared in vitro urea kinetics and digestion of forage-only or mixed forage-grain substrates in response to addition of NBPT. In vitro incubations were conducted in 50-mL test tubes containing either .5 g of ground fescue hay or .5 g of a ground fescue hay and ground corn mixture (50:50, DM basis) to which 0, 6.5, 13, 26, or 52 mg of NBPT in a buffered ruminal fluid innoculum was added. Tubes were incubated in triplicate at 39 degrees C and replicated on consecutive days, with NH3 N and urea concentrations measured at 0, .5, 1, 2, 4, 8, 12, 24, and 48 h. At 48 h, samples for VFA analysis were collected and true digestibility (based on NDF analysis) was estimated. No (P > .10) NBPT dose x substrate interactions were detected. Increasing the dose of NBPT depressed (P < .003) the rate of urea hydrolysis and subsequent NH3 N formation, regardless of substrate. Although total VFA concentration was unaffected (P > .10), the acetate:propionate and estimated true digestibility decreased (P < .002) with higher NBPT addition. In both experiments, the rate of urea degradation was not different (P > .20) from zero for the 26 and 52 mg NBPT treatments, indicating that nearly complete inhibition of urease had been achieved. We conclude that NBPT can be used to reduce the rate of NH3 N release from dietary urea and, thereby, offers the potential to improve nonprotein nitrogen utilization in ruminants.

Animal Feed↗

Simple routine assay for serum urea using immobilized urease.

The method described is a simple routine assay suited for a short series of serum samples. The time needed for one assay is 2 to 3 min from a stand-by arrangement. The urease is immobilized on controlled pore glass. The beads are placed in the column of an enzyme thermistor unit that is part of a continuous flow system. The heat of reaction when urea is degraded to ammonia and carbon dioxide by immobilized urease is measured and recorded continuously. The technique was investigated as regards to flow dependence, linearity, recovery, precision and some possible interfering substances. The within day precision was 0.8% (C.V.) and the day to day precision, during 56 days, was 3.0% (C.V.). Furthermore, the coefficient of correlation between results obtained with the enzyme thermistor unit and a conventional spectrophotometric method was 0.991.

Calorimetry↗

Biometric evaluation of gastric urease activity in man.

The 14C-urea breath test is claimed to be the best test for detection of Helicobacter pylori infection. We present a procedure for estimating its cutoff value on the basis of biometric evaluation only, without involving other diagnostic methods. Cumulative gastric urease activity (CA) was determined during the first 30 min after oral administration of 14C-urea (92.5 kBq) in 56 random volunteers and 49 consecutive patients with peptic ulcer disease. The distribution of CA in random volunteers was positively skewed. Logarithmic transformation yielded two separate populations, each normally distributed. Their normal probability density functions intercepted at log cutoff value. A bimodal distribution of log CA was confirmed in patients with peptic ulcer disease before and after treatment aimed at H. pylori eradication. Cutoff values were determined for both random volunteers and patients with peptic ulcer disease. By application of the present procedure for determination of cutoff value, the 14C-urea breath test distinguishes between individuals who have an increased gastric urease activity and individuals who do not, with the smallest possible arbitrariness.

Adult↗

A study of inhibition of urease from seeds of the water melon (Citrullus vulgaris).

Urease from seeds of the water melon was found to be inhibited by various salts of sodium. Sodium fluoride strongly inhibited the activity in the low urea concentration range. The enzyme was also inhibited by a high concentration of urea which was completely abolished in the presence of 10 mM sodium fluoride. Time-dependent inactivation of urease with iodoacetic acid, N-ethylmaleimide and p-hydroxymercuribenzoate exhibited biphasic kinetics in which half of the initial activity was lost in the fast phase and the remainder in a slow phase. Each phase exhibited first-order kinetics. These observations are suggestive of the existence of half-and-half distribution of sites.

Enzyme Activation↗

Effect of a urease inhibitor and ceiling fans on ascites in broilers. 1. Environmental variability and incidence of ascites.

A low ventilation model to induce ascites was introduced and characterized. In addition, the effect of supplemental air mixing via ceiling fans (CF) and the feeding of a urease inhibitor (0, 125, and 250 ppm) on incidence of ascites were investigated. Twelve environmental chambers were utilized in the trial; six were fitted with CF. Each dietary treatment was replicated twice per CF treatment. One hundred and twenty day-old male commercial broilers were reared per chamber. Atmospheric O2, CO2, and NH3, temperature, and humidity, as well as weekly litter moisture and pH, were monitored. Chamber CO2 levels increased immediately then stabilized. Chamber NH3 levels increased between 2 to 4 wk of age and rapidly declined when ventilation rates were increased to 1 cfm per bird. The CF and dietary treatments had little effect on air or litter variables except for NH3. Supplementing the diet with urease inhibitor resulted in a greater than 50% reduction in cumulative mortality due to ascites and a slight reduction in weekly BW gains. The CF treatment had no effect on production variables such as weekly feed intake, gain, and feed to gain ratio, or survivability due to ascites.

Ammonia↗

Diagnosis of Helicobacter pylori infection correlation between clo-test (urease enzyme) and gastric mucosal histology.

BACKGROUND: There are few histopathologists in West African region. Histology results on gastric mucosal biopsies for Helicobacter pylori (HP) usually take between one to two weeks to be out. On the other hand, clo-test results for Helicobacter pylori can be read between 5 minutes to 24 hours. Is one justified to commence therapy based on the clo-test ahead of the histology result? Where there is no histopathology services as in many centers in West Africa can one rely on clo-test alone? AIM: To evaluate the correlation between clo-test (urease) and histological findings of gastric mucosal biopsies. DESIGN: Prospective study. SETTING: Endoscopic unit of Obafemi Awolowo University Teaching Hospital Complex, Ile-Ife, Nigeria. PATIENTS: Twenty duodenal ulcer (DU) patients seen consecutively in the endoscopy unit. INTERVENTION: Upper gastrointestinal tract endoscopy, antral biopsies for the clo-test and histopathology. RESULT: There was 100% correlation between the clo-test and histological findings among all the 20 patients. CONCLUSION: Clo-test (urease) is reliable enough to be used to commence therapy aimed at ulcer healing and HP eradication.

Adult↗

Evaluation of culture, histological examination, serology and the rapid urease test for diagnosis of Helicobacter pylori in patients with dyspepsia in Bangladesh.

Helicobacter pylori, a gram-negative, microaerophilic bacterium, has been established to have a causal association with chronic gastritis, peptic ulcer, gastric adenocarcinoma, and low-grade lymphoma. The present study was undertaken to evaluate the efficacy of culture, histological examination, the rapid urease test, and serology for the diagnosis of H. pylori infection. A total of 45 consecutive subjects with various upper gastrointestinal symptoms were included in this study. The rates of diagnosis of H. pylori infection were 51.1%, 55.6%, 82.2%, and 93.3%, by culture, rapid urease test (RUT), histological examination, and serology, respectively. The sensitivity, specificity, and positive and negative predictive values were 95.5%, 82.6%, 84.0%, and 95.0%, respectively for RUT; 95.5%, 30.4%, 56.8%, and 87.5% for histological examination; 100%, 13.6%, 54.8% and 100% for serology.

Adult↗