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The kinetics of the inhibition of jack bean urease in the presence of ethylurea and N,N-dimethylurea was studied at pH = 7.0. Both inhibitors were competitive inhibitors. A simple novel method was used for determining the dissociation constants (Ki) values for the inhibition which were 26 and 28 mM respectively.
A simple novel method was introduced for determination of an inhibitor binding constant (Ki) and enthalpy of binding by isothermal titration microcalorimetry technique. This method was applied to the binding of fluoride ion, as an inhibitor, with the active sites of jack bean urease at pH = 7.0 (Tris 30 mM) and T = 300 degrees K. The dissociation equilibrium constant measured by this method was markedly consistent with the inhibition constant obtained from assay of enzyme activity in the presence of fluoride ion.
Seven species of human T-mycoplasmas that grow in Fraction A and 20 mug urea/ml died when the urea was omitted. Two species would not grow in Fraction A broth containing 10 mug/urea/ml. The other five strains grew in broth containing 10 mug urea/ml and were adapted by serial passage in broth containing decreasing concentrations of urea to grow in broth containing 2.5 mug/ml urea, but not in broth containing 1.25 mug/ml. Therefore the minimal urea requirement is not the same for the growth of all strains of T-mycoplasmas. In exponential phase broth cultures, urease was detected only intracellularly, none being found in the medium.
AIM: To prospectively assess the sensitivity, specificity and time to positivity of theUltra-rapid urease test (URUT) for Helicobacter pylori (H pylori), and compare the results of one with those of two biopsies. METHODS: Five antral biopsies were taken in consecutive patients undergoing upper endoscopy: one and two biopsies for URUT, and one each for H pylori culture and histology. URUT was read at 1, 5, 10, 20 and 30 min, 1, 2, 3 and 24 h after biopsy insertion into the reagent. A positive histology and/or culture was used as positive reference "gold standards". RESULTS: URUT was more sensitive for detecting H pylori with two biopsies rather than one, at all time points up to 120 min. The sensitivity improved from 3.6% to 82.1% for one biopsy and 10.7% to 85.7% for two biopsies from 1 to 120 min. The sensitivity reached 96.4% at 24 h for both, but the specificity reduced from 100% to 96% and 92% for one and two biopsies, respectively. CONCLUSION: Development of a positive URUT result is hastened by doubling the number of gastric biopsies. We recommend taking two instead of one biopsy to achieve an earlier positive URUT result so that H pylori eradication therapy can be initiated before patient is discharged from the endoscopy suite.
AIM: To evaluate whether attenuated Salmonella typhimurium producing Helicobacter pylori (H pylori) urease subunit B (UreB) could induce systemic immune responses against H pylori infection. METHODS: Attenuated S. typhimurium SL3261 was used as a live carrier of plasmid pTC01-UreB, which encodes recombinant H pylori UreB protein. Balb/c mice were given oral immunization with two doses of SL3261/pTC01-UreB at a 3-wk interval. Twelve weeks after oral immunization of mice, serum IgG antibodies were evaluated by ELISA assay. Gamma interferon (IFN-gamma) and interleukin 10 (IL-10) in the supernatant of spleen cell culture were also assessed by ELISA. RESULTS: After oral immunization of mice, serum specific IgG antibodies against UreB in vaccine group were much higher than that in PBS and native Salmonella SL3261 control groups (A450, 0.373+/-0.100 vs 0.053+/-0.022, 0.142+/-0.039, respectively, P<0.01). Moreover, IFN-gamma in vaccine group was on average 167.53+/-29.93 pg/mL, which showed a significant increase vs that of PBS control group (35.68+/-3.55 pg/mL, P<0.01). There was also a tremendous increase of IL-10 in vaccine group compared to PBS and SL3261 control groups (275.13+/-27.65 pg/mL vs 56.00+/-7.15 pg/mL, 68.02+/-15.03 pg/mL, respectively, P<0.01). In addition, no obvious side effects in mice and no change in gastric inflammation were observed. CONCLUSION: The multiple oral immunizations with the attenuated S. typhimurium expressing H pylori UreB could induce significant systemic immune responses, suggesting it may be used as oral vaccine against H pylori infection.
Rapid urease tests (RUTs) are simple and economical methods to detect Helicobacter pylori infection. However, they have been thought to be unreliable to confirm successful eradication of the infection following treatment. We reviewed the efficiency of RUTs and investigated their possible indication for use after eradication therapy. The sensitivity of highly sensitive RUTs seemed to be comparable to that of culture regardless of the timing for testing, but remarkably lower than that of histology within a few months after treatment. Highly sensitive RUTs, used several months after therapy, produced excellent accuracy, suggesting that they might be available to confirm successful treatment, taking cost and time factors into consideration.
AIM OF STUDY: To determine whether a homemade rapid urease test (RUT) (1 mL of 10% urea broth in distilled water plus one drop of 1% phenol red as indicator, cost/test USD0.19) was reliable when compared to histology in the diagnosis of HP infection. METHOD: Prospective consecutive sampling of patients who underwent outpatient oesophagogastroduodenoscopy and antral biopsies from October 1996 to January 1997. RUT and histology examinations were done on all specimens. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy of the RUT were calculated accordingly. RESULTS: Amongst our 140 patients, the sensitivity, specificity, positive and negative predictive values and accuracy of RUT were 94%, 99%, 99%, 95% and 96% respectively. Seventy-seven percent of the positive RUTs can be detected within 1 hour. CONCLUSION: Our homemade RUT is an inexpensive test with good sensitivity and specificity for HP infection.
Age-specific prevalence of IgA and IgG antibodies in 714 subjects without gastrointestinal complaints aged 6 months to 90 yr was measured by an enzyme linked immunoassay using an acid-glycine extract of H. pylori as the antigen. The urease test and histology were used for the diagnosis of H. pylori infection in 83 subjects with a clinical diagnosis of dyspepsia, and these results were compared with measurement of IgG, IgA and IgM antibodies. The age specific prevalence of IgG and IgA antibodies respectively was 57 and 43 per cent for subjects aged 6 months to 4 yr and showed an increase with age to a maximum of 90 per cent for IgG in subjects > 60 yr of age and to 87 per cent for IgA in subjects between 51 and 60 yr. In symptomatic patients, there was a high degree of correlation between severity of H. pylori infection on histopathological examination and IgG (P < 0.02) levels. The use of IgG and IgA estimation could have identified H. pylori infection without endoscopy in 50 of the 83 patients. Serology for IgG and IgA antibodies against H. pylori may play a major role in decreasing the need for endoscopy, but cut-off values must be determined for each assay based on the prevalence of antibodies in the population.
OBJECTIVE: To evaluate the immune prophylaxis of recombinant urease subunit A or B (rUreA or rUreB). METHODS: The BALB/C mice were divided into seven groups: negative control group, control group with CTB, control group with rUreA, control group with rUreB, experimental group with rUreA + CTB and experimental group with rUreB + CTB, experimental group with sonicate + CTB, which were gastrically inoculated with 150 ml PBS, 5 micrograms CTB, 50 micrograms rUreA, 50 micrograms rUreB, 50 micrograms rUreA + CTB, 5 micrograms rUreB + CTB and 400 micrograms sonicate + 5 micrograms CTB, respectively. Two weeks later, the mice were gastrically infected with H. pylori (10(7)-10(8) CPU per time). 4, 8 and 12 weeks later, the mice were killed and the immune protection of PBS, CTB, rUreA, rUreB, rUreA + CTB, rUreB + CTB and sonicate + CTB were evaluated by the presence of H. pylori in gastric tissue stained with Giemsa by microscopy. The mice in the side-effecting groups of rUreA + CTB and rUreB + CTB were not infected with H. pylori. 12 weeks later, the mice in the two groups were killed, and the side effects of rUreA + CTB and rUreB + CTB were evaluated by the infiltration of inflammation cells in gastric tissue stained with HE by microscopy. RESULTS: The mice gastrically inoculated with CTB, rUreA or rUreB had significantly less H. pylori. The immune protective effect of rUreA, rUreB or sonicate of H. pylori combined with CTB was stronger than that of rUreA, rUreB or sonicate of H. pylori alone. The immune protective effect of rUreA + CTB occurred later than that of rUreB + CTB or sonicate of H. pylori + CTB. The mice immunized with rUreA + CTB, rUreB + CTB or sonicate of H. pylori + CTB were not completely protected against H. pylori infection. The mice immunized with rUreA + CTB, rUreB + CTB or sonicate of H. pylori + CTB were less infected with H. pylori, and had mild inflammation of gastric tissue. CONCLUSION: Mice immunized with rUreA + CTB, rUreB + CTB or sonicate of H. pylori + CTB could not be completely protected against H. pylori infection and have alleviated inflammation of gastric tissue.
Pulsed-field gel electrophoresis (PFGE) analysis was carried out after separate digestion with Apa I, Sal I and Sma I of the genomic DNA from sixteen isolates of urease-positive thermophilic Campylobacter (UPTC) obtained from the natural environment, namely from oysters and mussels, in Northern Ireland. Five NCTC strains previously isolated in England were used for the analysis. Although the eight isolates of UPTC in Northern Ireland and a strain of UPTC in England showed that one or no fragments appeared after digestion with Apa I around 1,900 to 1,640 kb region of the gel, Apa I was shown to cut the genomic DNA from all of the other twelve strains of UPTC and Sal I and Sma I from all of the 21 strains in a distinctly different and distinguishable manner. Consequently, the present study clearly demonstrated that the sixteen isolates of UPTC in Northern Ireland and the five strains in England gave the diversity of the genomic DNA by using PFGE. Some strains of UPTC examined were shown to carry genomes from 1.6 to 1.9 Mb in length, thus the heterogeneous profiles of PFGE and the length of the genomes are thought to occur among the isolates of UPTC in Northern Ireland, as well as among the five representative strains of UPTC from NCTC.
OBJECTIVE: To establish attenuated Salmonella typhimurium producing Helicobacter pylori (H. pylori) urease subunit B (UreB) and determine whether it could be used as an oral vaccine against H. pylori. METHODS: H. pylori (SS1 strain) UreB gene fragment amplified by PCR was cloned into the prokaryotic expression vector pTC01 after sequencing, and then transformed into attenuated Salmonella typhimurium SL3261 to acquire SL3261/pTC01-UreB. The expression of H. pylori UreB in SL3261 was detected by Western blot. Twelve weeks after oral immunization of mice, antibody responses were evaluated using serum and intestinal fluid by ELISA assay. Interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) in the supernatant of spleen cells culture were also assessed by ELISA. In vitro stability of pTC01-UreB plasmid in SL3261 was confirmed by growing in Luria Broth (LB) medium to 80 generations. RESULTS: The UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB as evidenced by sequence analysis. Enzyme digestion revealed that the correct pTC01-UreB was obtained. Western blot showed that a 61kDa protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H. pylori UreB antiserum. After 80 generations of continuous culture, the recombinant plasmid pTC01-UreB was stable in SL3261 and had no obvious toxicity. Multiple oral immunizations with SL3261/pTC01-UreB could significantly induce H. pylori-specific mucosal IgA response as well as serum IgG response. Moreover, there were significant increases of IFN-gamma and IL-10 in the SL3261/pTC01-UreB group. Finally, no obvious side effects for mice and no change in gastric inflammation were observed. CONCLUSION: Attenuated Salmonella typhimurium expressing H. pylori UreB may be used as oral vaccine against H. pylori infection.
Today there are many methods in diagnostics of Helicobacter pylori infection. They are divided in two major groups based on using of endoscopy (invasive and non-invasive methods). Helicobacter pylori bacteria are specific because of having very big amounts of urease enzyme that divides urea on NH3 and CO2 which enables environment suitable for survival. Rapid ureas test is based on detecting of the enzyme activity. Because of its simplicity and high sensitivity and specificity it belongs to methods that are used in every day practice in endoscopic laboratories.
Rapid urease test(RUT) is one of the invasive methods for detection of H. pylori infection. H. pylori in the biopsy specimen splits the urea to ammonia in the test container. Elevation of pH by ammonium hydroxide are detected as color change of pH indicator. The advantage of RUT are low-priced, easy-to-use, and rapid time to diagnosis. The disadvantage of RUT are necessity of endoscopy performance and occurrence of false negative. RUT is useful for diagnosis of H. pylori infection before and after eradication treatment. However, the sensitivity of RUT is comparatively low within 6 months after completion of eradication.
OBJECTIVE: To perform genetic recombination of the urease B subunit (UreB) of Helicobacter pylori (Hp) and examine the biological properties of the recombinant protein. METHODS: The gene fragment encoding Hp UreB was isolated clinically from Chinese subjects by means of PCR, and cloned subsequently into an expression vector pET-11C-UreB for the non-fusion protein expression in E.coli BL21 (DE3) strain. RESULTS: The expression of recombinant UreB was achieved in E.coli BL21 with a relative molecular weight of approximately 62,000 at the expression ratio of 26%, and the first 15 amino acids of recombinant UreB were MKKISREYVSMYGP. The results of peptide mapping and amino acid compositional analysis were consistent with previous theoretical prediction, and enzyme-linked immunosorbent assay together with Western blotting indicated strong immunogenicity and reactivity of the recombinant protein in BalB/c mice, which were specifically recognized by polyclonal BalB/c mice anti-Hp sera or human sera infected with Hp. CONCLUSION: The results of this study has laid an solid immunological foundation for incorporating recombinant UreB as a subunit vaccine component against Hp.
A purification procedure has been developed by which urease activity in extracts from the cyanobacterium Anabaena cylindrica was enriched 500-fold. The procedure involves MgSO4 precipitation at 55 degrees C and chromatography on hydroxylapatite and diethylaminoethyl sephadex. Its molecular weight was measured by sedimentation equilibrium in an airfuge to be 197,000 +/- 2000 with an estimated subunit molecular weight of 32,000 as determined by polyacrylamide gel electrophoresis. The pH- and temperature-dependence of the enzyme were determined and the activity found to be optimal at pH 8 and 30 degrees C, respectively. The concentration-dependence of the activation of the enzyme by Mg++ was measured, as were the effects on activity of a range of other metal ions.
UNLABELLED: The aim of our study was to develop a rapid diagnostic urease test to demonstrate the presence of Helicobacter pylori in the Endoscopy room. MATERIALS AND METHODS: 200 consecutive patients referred to gastroscopy for different indications, were included in this study. One antral biopsy sample was obtained to be immersed in our test. The same sample was used for histological evaluation, considered to be the gold standard method for diagnose of Helicobacter pylori infection. RESULTS: 135 patients (67.5%) were found positives and 65 patients (32.5%) were negatives in our test. 128 patients (64%) showed Helicobacter pylori on histological examination. Our test showed a sensitivity of 91%, specificity of 88.1%, and positive and negative predictive values of 95% and 80% respectively. A remarkable correlation between density of Helicobacter pylori and reading time was also observed, where a high density of the bacteria reduced the reaction time in this liquid test. Furthermore, an overall accuracy of 90% was shown, which is comparable with other available commercial tests. CONCLUSION: LUT is easy to handle, cost effective and fast, with a high positive predictive value.
AIM: To observe the immunopotency of the recombinant urease B subunit (rUreB) of Helicobacter pylori after intranasal administration to mice. MDTHODS: BALB/c mice were immunized intranasally with rUreB of 20 microg,10 microg and rUreB plus different adjuvants, such as cholera toxin B subunit (CTB), Escherichia coli heat labile enterotoxin B subunit (LTB) and carbopol respectively, four times at an intervals of 7 days. The serum and washing solution from gastric, intestinal, nasal and tracheal mucosas were collected in 7 days after final immunization. IgG and IgA antibodies specific for rUreB were detected by ELISA. RESULTS: The levels of IgA and IgG antibodies in sera every groups of mice immunized intranasally were significantly increased compared with control group (P<0.01). Only the levels of serum IgG of mice immunized with 20 microg dose were higher than those of mice immunized with 10 microg dose. Carbopol could enhance the level of IgA antibodies in washing solution from mouse gastric mucosa after intranasal immunization. The efficacy of LTB as a nasal mucosal immuno-adjuvant was stronger than that of CTB. CONCLUSION: CTB, LTB and carbopol can play the role of adjuvant in nasal mucosal immunization. Intranasal immunization with rUreB can induce not only serum IgG antibody production but also antibody responses of different mucosa. Thus intranasal inoculation is a convenient, effective and cheap immunization way.
With simulation test of in-situ soil column, this paper studied the effects of urease inhibitor hydroquinone (HQ), nitrification inhibitors coated calcium carbide (ECC) and dicyandiamide (DCD),and their different combinations on the persistence, oxidation, and leaching of soil urea's hydrolyzed product ammonium. The results showed that compared with other treatments, the combination of HQ and DCD could effectively inhibit the oxidation of the ammonium, and make it as exchangeable form reserve in soil in a larger amount and a longer period. The inhibition of this oxidation not only decreased the accumulation of oxidized product NO3- in soil, but also decreased the potential of NO3- leaching, making the NO3- only leach to 5-10 cm in depth, and the leached amount significantly decreased.