Intracellular Vacuolization Caused by the Urease of Helicobacter pylori
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Tissue culture cells were exposed to supernatants of Helicobacter pylori for 24 h at 37 degrees C in the presence of various quantities of urea. In the normal human stomach the concentration of urea is less than or equal to 4 mmol/l, and in the presence of this low concentration up to 10% of Vero cells showed intracellular vacuolization. In the presence of 7.5 mmol/l urea, 25% of the cells showed vacuolization. With 30 mmol/l urea, the final pH was 7.6, indicating that vacuolization was not due to change of pH. The first report of vacuolization of tissue culture cells by H. pylori was in a system without added urea but with concentrated bacterial supernatant; 30% of H. pylori strains demonstrated a cytotoxic effect. In those experiments fetal calf serum was used; it contains 6 mmol/l urea but was used at a concentration of 10%. A urease inhibitor, acetohydroxamic acid, caused a 75% drop in the number of cells showing vacuolization, and ammonia caused vacuolization. Thus the urea of H. pylori probably causes this vacuolization.Keywords:
Vacuolization
ABSTRACT Work in the United States of America has shown that dietary supplements of nickel (Ni) can result in an increase in rumen urease activity and can increase growth rate and food conversion efficiency in lambs and steers given low protein diets. It has been suggested that Ni enhances urea recycling through its effect on urease activity. To examine this hypothesis, four sheep were given a high-energy, lowprotein diet with or without a supplement of 5 mg/day Ni, given as NiCl2.6H2O by continuous infusion into the rumen. Urea irreversible loss rate (ILR) and pool size were measured by means of a single intravenous injection of 14C-urea. The addition of Ni resulted in a significant increase in rumen urease activity ( P < 0·05) but there was no significant effect on urea ILR, urea pool size, urinary urea excretion or on the quantity of urea recycled to the gastro-intestinal tract. Ni also had no effect on plasma urea or rumen ammonia concentrations, on diet digestibility or on nitrogen retention. It is concluded that the enhancement of urease activity by supplementary Ni conferred no nutritional advantage to the sheep.
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For decomposition of urea in sake, urease having activity at acidic side was studied.The optimum pH of the urease was 4.5-5.5, and it lost the activity perfectly by heating at 75°C for 15 min.Amounts of urea decomposed were proportional to temperature till 25°C, and also enzyme concentration and urea concentration.In order to obtain over 90% of urea decomposition rate in sake, it took 14 days at 10°C, and 3 days at 20°C by using enzyme concentration of 5 U/g.
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Recent work in our laboratory showed that the adverse effect of urea fertilizer on seed germination and seedling growth in soil is due to ammonia produced through hydrolysis of urea by soil urease (NH 2 CONH 2 + H 2 O → 2NH 3 + CO 2 ) and can be eliminated by amending the fertilizer with a small amount of a urease inhibitor such as phenylphosphorodiamidate. Because the leaf-tip necrosis often observed after foliar fertilization of plants with urea is usually attributed to ammonia formed through hydrolysis of urea by plant urease, we studied the possibility that this necrosis could be eliminated or reduced by adding phenylphosphorodiamidate to the urea fertilizer. We found that, although addition of this urease inhibitor to foliar-applied urea increased the urea content and decreased the ammonia content and urease activity of soybean [ Glycine max. (L.) Merr.] leaves fertilized with urea, it increased the leaf-tip necrosis observed after fertilization. We conclude that this necrosis resulted from accumulation of toxic amounts of urea rather than from formation of toxic amounts of ammonia. This conclusion was supported by our finding that the necrotic areas of soybean leaves treated with urea or with urea and phenylphosphorodiamidate contained much higher concentrations of urea than did the nonnecrotic areas.
Phytotoxicity
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SUMMARY: Urease or ATP: urea amidolyase activity was detected in extracts of unicellular algae (representing two phyla and five classes) grown with urea as sole source of nitrogen. All the algae examined that are unequivocally classified as Chloro-phyceae, contained ATP:urea amidolyase but no urease. All the other algae contained urease but no ATP: urea amidolyase.
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In studying the mechanism of urease action, it would be of interest to find a derivative of urea that would be hydrolyzed in the presence of urease. Urease is very specific in its action and urea appears to be its only substrate. Closely related compounds such as amides or purines as well as simple derivatives of urea are not attacked by this enzyme. Armstrong and Horton found that substitution in the urea molecule with methyl or ethyl groups invariably rendered the substituted urea inaccessible to urease. Schoorl synthesized glucose-urea, in which one molecule of urea was united with one molecule of the sugar. This compound is very soluble in water and is stable in solution. When heated with acid it undergoes hydrolysis with the formation of -glucose and urea. Johnson and Bergmann, in their recent researches on nitrogenous glucosides have prepared glucose urea, and Dr. Johnson generously placed at our disposal a very pure sample of glucose urea for investigation. We have studied the action of urease on this urea derivative. Jack bean urease was found to have no ability to decompose glucose urea. There was no ammonia production in 10 minutes when urease was added to 0.2 M solutions of this compound. However, if glucose-urea was hydrolyzed by acid prior to the addition of urease, ammonia production occurred at a rate comparable to that observed when a 0.2 M solution of urea and glucose was exposed to urease. The results (Table I) clearly demonstrate that the inability of urease to split glucose urea must be ascribed to the chemical make-up of the compound and cannot be the result of the presence, in the preparation, of substances that inactivate the enzyme.
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Soil incubation under 25℃ was applied to study the ammonia volatilization and soil urease activity of normal urea and value-added urea which was manufactured by melted urea with different organic synergists.The results showed that the value-added urea F-5 and H-5 decreased the accumulation of ammonia volatilization by 15.2% and 13.3% compared to the normal urea.The peak of ammonia volatilization was deferred by the treatment F-5 and the peak value was brought down by the two treatments whose variation curves were relatively stable.The treatment F-5 and H-5 could inhibit the soil urease activity and prolong the release time of urea.Compared with normal urea,the effects of F and H value-added urea on the ammonia volatilization reduction and the soil urease activity inhibition were significant.
Volatilisation
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A urea electrode using an ammonium ion-selective electrode with an immobilised urease membrane is described. The measurement of blood urea was carried out accurately without any pre-treatment of the samples. A conversion equation related to haematocrit was derived, which made it possible to determine plasma urea concentration from whole blood urea concentration. Rapid determinations of plasma urea concentrations were also made possible.
Ion selective electrode
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Eradication of Helicobacter pylori with currently available antibacterial agents is unsatisfactory due to the risk of side-effects and the emergence of resistance. The organism rapidly dies in vitro in the presence of urea at pH 6. When incubated in citrate buffer (pH 6) plus urea (10 mM) the five minute survival was 26% compared with 96% without urea and the survival progressively decreased with increasing urea concentrations, being only 9% in 50 mM urea. The bactericidal effect depended on pH as the organism survived in citrate buffer (pH 7) plus urea (50 mM). The death of the organism at pH 6 in the presence of urea was prevented by the addition of the competitive urease inhibitor hydroxyurea. These findings indicate that destruction of the organism is mediated by its exceptionally high urease activity. Harnessing this enzyme to induce self-destruction could provide a new approach to eradicating this common infection.
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