Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.3.4.6 (urease)
7,490 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To produce bispecific antibodies (BiAbs) for enzyme immunoassay (EIA) to replace antibody-enzyme conjugates, we developed a panel of 8-azaguanine/ouabain-resistant anti-urease variant hybridoma cell lines for use in hybridoma-hybridoma fusions. These variants represent mouse immunoglobulin subclasses IgM, IgG1, IgG2a, and IgG2b and have growth rates equal to those of the parental hybridomas. We fused an anti-urease-secreting variant hybridoma with an anti-human choriogonadotropin (hCG)-secreting hybridoma (both of IgG1 subclass) and selected the desired product with growth media containing hypoxanthine-aminopterin-thymidine (HAT) and ouabain. Over 95% of the resulting hybrids secreted anti-urease, and 60% of these secreted anti-hCG. The bispecific nature of secreted antibodies was demonstrated in a simultaneous EIA where BiAbs, hCG, and urease (EC 3.5.1.5) were incubated together in anti-hCG-coated microwells. As little as 25 int. units of hCG per liter could be reliably detected, which is equivalent to that for antibody-enzyme conjugates in EIA.
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PMID:Production of murine hybrid-hybridomas secreting bispecific monoclonal antibodies for use in urease-based immunoassays. 245

An in vitro stimulation protocol has been established which allows production of IgG-secreting murine hybridomas. This procedure has been examined using jack bean urease and human luteinizing hormone as antigens. Parameters which have been optimized include selection of media and serum supplements, thymocyte-conditioned media, antigen dosage, length of stimulation and the effect of medium changes during stimulation and additions of polyclonal mitogen. Murine spleen cells (1 X 10(8) in 10 ml) were incubated with varying doses of jack bean urease and human luteinizing hormone in a six-well plate in supplemented DMEM with 5% normal rabbit serum and 10% thymocyte-conditioned media. Following 5 and/or 8 days stimulation, the spleen cells were fused with SP2/0 cells and plated in 96-well plates. Stable hybridomas were obtained for both antigens from over 25% of the wells identified in initial screening for specific antibody production. All monoclonal antibodies obtained in the LH stimulation experiments, with one exception, were of the IgM isotype. A large number of IgG-producing hybridomas were isolated following prolonged (8 day) stimulation with high concentrations of urease, during which time the medium remained unchanged. Addition of polyclonal mitogen (E. coli lipopolysaccharide) at 10 micrograms/ml markedly increased the production of hybridomas secreting anti-urease, but most were of IgM class.
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PMID:Production of IgG-producing hybridomas by in vitro stimulation of murine spleen cells. 349 63

Acetohydroxamic acid (AHA), a potent urease inhibitor used for treatment of infection-induced struvite urolithiasis, was teratogenic after administration of 25 mg of AHA/kg of body weight/day orally to 5 clinically normal Beagles from the onset of proestrus until parturition. Thirty pups exposed to AHA in utero developed anomalies of the skeletal system, heart, and ventral midline. Cardiac anomalies included atrial septal defects (20%), ventricular septal defects (3%), and atrial and ventricular septal defects (3%). Skeletal anomalies included coccygeal hemivertebrae and fused coccygeal vertebrae (50%), supernumerary vertebrae (67%), supernumerary ribs (50%), duplicated sternebrae (3%), and lumbar hemivertebrae (3%). Defects of the ventral midline of the abdominal wall occurred in 20% of AHA-exposed pups. Other abnormalities included retarded growth, high neonatal mortality, and a decreased number of circulating RBC, compared with those in 30 control pups born to 5 Beagles given a placebo. Adverse effects of AHA in pregnant Beagles were limited to morphologic alterations (Howell-Jolly bodies, spherocytes, and target cells) in a small number of circulating RBC. Slight neutrophilic leukocytosis and monocytosis occurred between 0 and 30 days of pregnancy in dogs given AHA, compared with those in controls. Seemingly, AHA did not influence fertility, conception rate, or length of gestation.
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PMID:Teratogenic effect of acetohydroxamic acid in clinically normal beagles. 380 Jan 19

All Helicobacter pylori isolates synthesize a 54 kDa immunodominant protein that was reported to be associated with the nickel-dependent urease of H. pylori. This protein was recently recognized as a homologue of the heat-shock protein of the GroEL class. The gene encoding the GroEL-like protein of H. pylori (HspB) was cloned (pILL689) and was shown to belong to a bicistronic operon including the hspA and hspB genes. In Escherichia coli, the constitutive expression of the hspA and hspB genes was initiated from a promoter located within an IS5 insertion element that mapped upstream to the two open reading frames (ORFs). IS5 was absent from the H. pylori genome, and was thus acquired during the cosmid cloning process. hspA and hspB encoded polypeptides of 118 and 545 amino acid residues, corresponding to calculated molecular masses of 13.0 and 58.2 kDa, respectively. Amino acid sequence comparison studies revealed that, although H. pylori HspA and HspB proteins were highly similar to their bacterial homologues, the H. pylori HspA featured a striking motif at the C-terminus. This unique motif consists of a series of cysteine and histidine residues resembling a nickel-binding domain, which is not present in any of the other bacterial GroES homologues so far characterized. When the pILL689 recombinant plasmid was introduced together with the H. pylori urease gene cluster (pILL763) into an E. coli host strain, an increase of urease activity was observed. This suggested a close interaction between the HspA and HspB proteins and the urease enzyme, and a possible role for HspA in the chelation of nickel ions. The genes encoding each of the HspA and HspB polypeptides were cloned, expressed independently as proteins fused to the maltose-binding protein (MBP) and purified in large scale. The MBP-HspA and MBP-HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H. pylori-infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic.
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PMID:Helicobacter pylori hspA-hspB heat-shock gene cluster: nucleotide sequence, expression, putative function and immunogenicity. 771 57

Helicobacter pylori synthesizes a heat-shock protein of the GroES class. The gene encoding this protein (heat-shock protein A, HspA) was recently cloned and it was shown to be unique in structure. H. pylori HspA consists of two domains: the N-terminal domain (domain A) homologous with other GroES proteins, and a C-terminal domain (domain B) corresponding to 27 additional residues resembling a metal-binding domain. Various recombinant proteins consisting of the entire HspA polypeptide, the A domain, or the B domain were produced independently as proteins fused to maltose-binding protein (MBP). Comparison of the divalent cation binding properties of the various MBP and MBP-fused proteins allowed us to conclude that HspA binds nickel ions by means of its C-terminal domain. HspA exhibited a high and specific affinity for nickel ions in comparison with its affinity for other divalent cations (copper, zinc, cobalt). Equilibrium dialysis experiments revealed that MBP-HspA binds nickel ions with an apparent dissociation constant (Kd) of 1.8 microM and a stoichiometry of 1.9 ions per molecule. The analysis of the deduced HspA amino acid sequences encoded by 35 independent clinical isolates demonstrated the existence of two molecular variants of HspA, i.e. a major and a minor variant present in 89% and 11% of strains, respectively. The two variants differed from each other by the simultaneous substitution of seven amino acids within the B domain, whilst the A domain was highly conserved amongst all the HspA proteins (99-100% identity). On the basis of serological studies, the highly conserved A domain of HspA was found to be the immunodominant domain. Functional complementation experiments were performed to test the properties of the two HspA variants. When co-expressed together with the H. pylori urease gene cluster in Escherichia coli cells, the two HspA variant-encoding genes led to a fourfold increase in urease activity, demonstrating that HspA in H. pylori has a specialized function with regard to the nickel metalloenzyme urease.
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PMID:Nickel binding and immunological properties of the C-terminal domain of the Helicobacter pylori GroES homologue (HspA). 897 21

Bordetella bronchiseptica is a common ureolytic mammalian respiratory pathogen. The urease operon of this organism is encoded within an 8.9 kb DNA fragment which contains the structural genes (ureA, ureB and ureC) and accessory genes (ureD and ureG) homologous to other urease genes. Uniquely, the ureE and ureF genes are fused to form a hybrid protein, UreEF, which may result in tighter coordination of the putative functions of the individual accessory genes, nickel donation to the urease active site, and prevention of nickel incorporation until correct formation of the active site, respectively. The operon contains an additional open reading frame, UreJ, found only also in the Alcaligenes eutrophus urease operon. UreJ is also 37% homologous with HupE from Rhizobium leguminosarum bv. viciae, and may potentially be involved in nickel transport. A transcriptional activator, designated Bordetella bronchiseptica urease regulator (BbuR), is located directly upstream and in the opposite orientation to the urease operon. BbuR shares homology with members of the LysR regulatory protein family. LysR proteins have been shown to regulate urease in Klebsiella aerogenes (NAC), and catalase in Escherichia coli (OxyR), which offers the intracellular bacterium protection from phagolysosome damage. A putative BbuR binding site (5'-ATA-N9-TAT-3'), identical to the NAC-binding consensus sequence, was found 27 bp upstream of the urease promoter in B. bronchiseptica. We hypothesise that BbuR controls urease expression which is involved in protection of intracellular B. bronchiseptica from phagolysosomal damage. Comparison of the urease promoter regions of B. bronchiseptica, Bordetella parapertussis ATCC15311 and the urease-negative strain B. pertussis Tohama I revealed no differences in the ureD open reading frame between each species. A cluster of mutations in both B. pertussis and B. parapertussis was found upstream of the urease promoter, in a region proximal to the putative bbuR promoter. The inability of B. pertussis to produce urease may therefore reflect mutations in regulatory elements, and not mutations in the urease locus itself.
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PMID:Characterisation of the urease gene cluster in Bordetella bronchiseptica. 952 76

Complex metalloenzymes (e.g., nitrogenase, hydrogenase, urease) are synthesized starting from the apoprotein via several intermediates by the action of accessory proteins. The isolation and biochemical characterization of such intermediates is hampered by their low abundance and their lability. Here we describe a technique for efficient single-step purification of a hydrogenase precursor under mild conditions using a N-terminal Strep-tag II affinity peptide and a novel StrepTactin Sepharose matrix. The tag was fused to the large subunit of [NiFe] hydrogenase 3 (HycE) of Escherichia coli. No significant influence of the affinity peptide on maturation or activity of the protein was observed when the modified gene was integrated into the chromosome by homologous recombination. A tagged nickel-free precursor form of HycE bound quantitatively to a recombinant StrepTactin Sepharose column. More than 90% pure subunit could be obtained after elution with desthiobiotin. The procedure was shown to be more efficient than purification by immobilized metal affinity chromatography using a N-terminal His-tag. General advantages of the novel Strep-tag II affinity purification especially for applications with metalloenzymes are discussed.
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PMID:Strep-tag II affinity purification: an approach to study intermediates of metalloenzyme biosynthesis. 960 45

We describe a new procedure allowing the generation and detection of immunogenic antigens from Helicobacter pylori via the hemolysin secretion apparatus of Escherichia coli. The gene (or gene fragment) encoding the H. pylori protein (or protein domain) is inserted in-frame into a residual portion of the hemolysin gene (hlyA), encoding the HlyA secretion signal (HlyA(s)). These fusion proteins are secreted efficiently by E. coli. This new approach allows the identification of immunodominant antigens by using sera derived from H. pylori-infected patients suffering from different gastroduodenal pathologies. Three immunodominant antigens bearing the ureB (urease B-subunit), flaA (flagellin A-subunit), and an unknown ORF (HP0888) encoding an E. coli FecE analogous protein fused to hlyA(s) were identified and characterized.
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PMID:Identification of immunogenic antigens of Helicobacter pylori via the Escherichia coli hemolysin secretion system(1). 1080 80

Analysis of a Brucella suis 1330 gene fused to a gfp reporter, and identified as being induced in J774 murine macrophage-like cells, allowed the isolation of a gene homologous to nikA, the first gene of the Escherichia coli operon encoding the specific transport system for nickel. DNA sequence analysis of the corresponding B. suis nik locus showed that it was highly similar to that of E. coli except for localization of the nikR regulatory gene, which lies upstream from the structural nikABCDE genes and in the opposite orientation. Protein sequence comparisons suggested that the deduced nikABCDE gene products belong to a periplasmic binding protein-dependent transport system. The nikA promoter-gfp fusion was activated in vitro by low oxygen tension and metal ion deficiency and was repressed by NiCl(2) excess. Insertional inactivation of nikA strongly reduced the activity of the nickel metalloenzyme urease, which was restored by addition of a nickel excess. Moreover, the nikA mutant of B. suis was functionally complemented with the E. coli nik gene cluster, leading to the recovery of urease activity. Reciprocally, an E. coli strain harboring a deleted nik operon recovered hydrogenase activity by heterologous complementation with the B. suis nik locus. Taking into account these results, we propose that the nik locus of B. suis encodes a nickel transport system. The results further suggest that nickel could enter B. suis via other transport systems. Intracellular growth rates of the B. suis wild-type and nikA mutant strains in human monocytes were similar, indicating that nikA was not essential for this step of infection. We discuss a possible role of nickel transport in maintaining enzymatic activities which could be crucial for survival of the bacteria under the environmental conditions encountered within the host.
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PMID:Identification of the nik gene cluster of Brucella suis: regulation and contribution to urease activity. 1113 34

The metabolism of urea by urease enzymes of oral bacteria profoundly influences oral biofilm pH homeostasis and oral microbial ecology. The purpose of this study was to gain insight into the regulation of expression of the low pH-inducible urease genes in populations of Streptococcus salivarius growing in vitro in biofilms and to explore whether urease regulation or the levels of urease expression in biofilm cells differed significantly from planktonic cells. Two strains of S. salivarius harbouring urease promoter fusions to a chloramphenicol acetyltransferase (cat) gene were used: PurelCAT, containing a fusion to the full-length, pH-sensitive promoter; or Pureldelta100CAT, a constitutively derepressed deletion derivative of the urease gene promoter. The strains were grown in a Rototorque biofilm reactor in a tryptone-yeast extract-sucrose medium with or without pH control. Both CAT and urease activities in biofilms were measured at 'quasi-steady state' and after a 25mM glucose pulse. The results showed that CAT expression in PurelCAT was repressed at relatively neutral pH values, and that expression could be induced by acidic pH after carbohydrate challenge. Biofilms of PurelCAT grown at low pH, without buffering, had about 20-fold higher CAT levels, and only a modest further induction could be elicited with carbohydrate pulsing. The levels of CAT in biofilms of PurelCAT grown in buffered medium were slightly higher than those reported for planktonic cells cultured at pH 7.0, and the levels of CAT in Purel-CAT growing at low pH or after induction were similar to those reported for fully induced planktonic cells. CAT activity in Pureldelta100CAT was constitutively high, regardless of growth conditions. Interestingly, urease activity detected in biofilms of the parent strain, S. salivarius 57.1, could be as much as 130-fold higher than that reported for fluid chemostat cultures grown under similar conditions. The higher level of urease activity in biofilms was probably caused by the accumulation of the stable urease enzyme within biofilm cells, low pH microenvironments and the growth phase of populations of cells in the biofilm. The ability of S. salivarius biofilm cells to upregulate urease expression in response to pH gradients and to accumulate greater quantities of urease enzyme when growing in biofilms may have a significant impact on oral biofilm pH homeostasis and microbial ecology in vivo. Additionally, S. salivarius carrying the pH-sensitive urease gene promoter fused to an appropriate reporter gene may be a useful biological probe for sensing biofilm pH in situ.
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PMID:Regulation of urease gene expression by Streptococcus salivarius growing in biofilms. 1122 Mar 3


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