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:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An enzyme-linked immunosorbent assay for the detection and quantitation of human immunoglobulin M (IgM), IgG, and IgA antibodies against Yersinia enterocolitica is described. Formalinized or heat-treated bacteria were adsorbed onto specially designed microcuvettes, and antibodies were allowed to attach to the antigen-coated cuvettes. Rabbit anti-human mu, anti-human gamma, and anti-human alpha antisera were allowed to react with human antibodies, and these class-specific anti-immunoglobulins were detected by alkaline phosphatase-labeled swine anti-rabbit IgG. A total of 423 sera were tested. The results obtained with the enzyme-linked immunosorbent assay were compared with the results of the conventional tube agglutination test. Persistence of different antibodies was studied in six patients. Antibodies of the IgM class persisted only for 1 to 3 months after onset of the disease; thus the occurence of IgM-class Yersinia antibodies in a single sample indicates a recently acquired infection. The persistence of the IgG- and IgA-class antibodies was variable and not parallel with each other. Remarkably, all three patients in which the disease was complicated with arthritis had IgA-class Yersinia antibodies at the end of the follow-up period of 9 to 14 months, and in those without arthritis the IgA-class antibodies disappeared within 3 months after onset of the disease.
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PMID:Measurement of immunoglobulin M (IgM), IgG, and IgA antibodies against Yersinia enterocolitica by enzyme-linked immunosorbent assay: persistence of serum antibodies during disease. 37 30

Two phenotypes believed to contribute to the pathogenesis of Salmonella infections are macrophage survival and invasion of epithelial cells. It was recently observed that the Salmonella macrophage survival factor PagC has significant amino acid similarity to the Yersinia invasion factor Ail. This observation raised the possibilities that macrophage survival is in part determined by the pathway of entry and that PagC confers an entry mechanism that does not trigger the microbicidal activities of the macrophage. Thus, we sought to investigate the role of PagC in invasion by examining (i) the invasion phenotype of pagC mutants and (ii) the invasion phenotype of Escherichia coli carrying pagC. A previously identified invasion-defective TnphoA insertion mutant of Salmonella enteritidis was found to have TnphoA inserted into the signal sequence-encoding region of pagC; the pagC allele from this mutant, SM5T, was designated pagC64. In contrast, Salmonella typhimurium carrying the pagC1 allele (a TnphoA insertion mutation, downstream of the region encoding the signal sequence) was not defective for invasion. Further analysis of these two pagC alleles suggested that the invasion-defective phenotype associated with pagC64 is not due to the loss of PagC function but rather is due to the synthesis of a hybrid PagC-alkaline phosphatase protein that is aberrantly localized, most likely to the inner membrane, and thus may prevent proper localization or function of a factor(s) required for efficient invasion. The observation that pagC did not confer an invasive phenotype to E. coli further suggests that PagC is not an invasion factor. A cloned pagC gene complemented the macrophage survival defect of S. typhimurium pagC1 mutants, but the cloned ail gene did not. Together these results suggest that the structural similarity between PagC and Ail may not extend to a similarity in function. Interestingly, S. enteritidis carrying the pagC64 allele that results in both an invasion defect and a macrophage survival defect was less virulent for mice infected intragastrically or intraperitoneally than was S. enteritidis carrying the pagC1 allele that results only in a macrophage survival defect.
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PMID:An unusual pagC::TnphoA mutation leads to an invasion- and virulence-defective phenotype in Salmonellae. 132 35

The enzymic activity (succinate dehydrogenase, acid and alkaline phosphatase) of alveolar and peritoneal macrophages as well as leucocytic reaction of ground squirrels infected with Yersinia pseudotuberculosis (I and III serovar) have been investigated in dynamics from the 1st up to the 30th day. The animals infected with III serovar survived only to the 7th day, while those infected with I serovar survived up to the 30th day after inoculation. A massive influx of leucocytes having peak values (100-fold increase) on the 3rd day after infection has been found in the peritoneal cavity of the animals infected with I serovar. Moderate leucocytosis in the blood, and insignificant fluctuations in alveolar macrophage number have been established too. An earlier and higher activation of succinate dehydrogenase in alveolar and peritoneal macrophages from animals infected with III serovar in comparison with those infected with I serovar was observed. No differences in alkaline phosphatase activity of alveolar and peritoneal macrophages have been found between the animals infected with I and III serovar. A correlation has been found between the number of leucocytes and changes in the enzymatic activity of the macrophages. A metabolic transformation was demonstrated typical for different macrophages (peritoneal and alveolar), in the course of this experimental intraperitoneal infection. Obviously, more virulent serovar III of Y. pseudotuberculosis fails to attract leucocytes to the peritoneal cavity sufficiently quickly, so it overcome the local protective mechanisms with consequent systemic cytochemical changes. On the other hand the virulent serovar I attracts leucocytes to the peritoneum and is presumably destroyed by them.
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PMID:Cytometric and cytochemical study of peritoneal and alveolar macrophages from Yersinia pseudotuberculosis infected ground squirrels (Citellus citellus). 149 17

Yersinia pestis, the etiologic agent of bubonic plague, contains a 75-kb virulence plasmid, called pCD1 in Y. pestis KIM. The low-Ca(2+)-response genes of Y. pestis regulate both bacterial growth and the expression of pCD1-encoded virulence determinants in response to temperature and the presence of Ca2+ or nucleotides. This study characterizes the nucleotide sequence and protein product of the lcrD locus. An lcrD mutant, in contrast to the parent Y. pestis, did not undergo growth restriction or induce strong expression of the V antigen when grown under conditions (37 degrees C, no Ca2+) expected to elicit maximal expression of pCD1 genes. DNA sequence analysis of the cloned lcrD locus showed a single open reading frame that could encode a protein with a molecular weight of 77,804 and a pI of 4.88. LcrD was identified as a 70-kDa inner membrane protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. LcrD membrane topology was investigated by using lcrD-phoA translational fusions generated with the transposon TnphoA. The alkaline phosphatase activities of the resultant hybrid proteins were consistent with a model predicting eight amino-terminal transmembrane segments that anchor a large cytoplasmic carboxyl-terminal domain to the inner membrane.
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PMID:LcrD, a membrane-bound regulator of the Yersinia pestis low-calcium response. 165 87

After incubation at 37 degrees C in the absence of Ca2+ ions, pathogenic strains of Yersinia spp. release large amounts of a set of plasmid-encoded proteins called Yops. The secretion of these proteins, involved in pathogenicity, occurs via a mechanism that involves neither the removal of a signal sequence nor the recognition of a C-terminal domain. Analysis of deletion mutants allowed the secretion recognition domain to be localized within the 48 N-terminal amino acids of protein YopH, within the 98 N-terminal residues of protein YopE, and within the 76 N-terminal residues of YopQ. Comparison of these regions failed to reveal any sequence similarity, suggesting that the secretion signal of Yop proteins is conformational rather than sequential. Hybrid proteins containing the amino-terminal part of YopH fused to either the alpha-peptide of beta-galactosidase or to alkaline phosphatase deprived of its signal sequence were efficiently secreted to the Yersinia culture medium. This observation opens new prospects in using Yersinia spp. as chimeric-protein producers and as potential live carriers for foreign antigens.
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PMID:Secretion of hybrid proteins by the Yersinia Yop export system. 199 87

We established the enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to Campylobacter and applied it in defining the period of the primary infection of Campylobacter in infant cynomolgus monkeys (Macaca fascicularis). The antibody to Campylobacter spp. could be detected with only 0.25 mul of serum by using commercially available antigens and anti-cynomolgus monkey IgG antibody conjugated with alkaline phosphatase. The inhibition experiments using extracts of C. jejuni, C. fetus and Yersinia enterocolitica demonstrated that the established ELISA system could detect species-specific anti-C. jejuni and anti-C. fetus antibodies. The levels of antibodies to both C. jejuni and C. fetus were high in 2 weeks old infant cynomolgus monkeys, rapidly decreasing until 6 to 14 weeks of age. This result indicates that the antibodies detected in 2 week old infants were IgG antibodies of maternal origin transferred through placenta. The C. jejuni was isolated from infants when the level of maternal antibody became the lowest. Infant cynomolgus monkeys obviously developed IgG antibodies to C. jejuni within 4 weeks after infection. On the other hand, no antibody response to C. jejuni was found in two infants from which it could not be isolated throughout the observation period. As regards C. fetus infection, infants showed a poor antibody response although it was more frequently isolated than C. jejuni. In conclusion, the ELISA system established in the present study is useful for the serological diagnosis of C. jejuni infection during infancy in the cynomolgus monkey.
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PMID:The serodiagnosis of Campylobacter infection in infant cynomolgus monkeys (Macaca fascicularis) 2 to 18 weeks old by enzyme-linked immunosorbent assay. 322 62

An enzyme immunoassay (EIA) was developed for the detection of Yersinia-immunoglobulin complexes of known Ig class. Immune complexes (ICs) were attached to polystyrene microtiter plates by rabbit anti-human immunoglobulins, and the existence of Yersinia enterocolitica O:3 antigens was demonstrated using Fab fragments of alkaline phosphatase (AP)-conjugated antibody against the same serotype. Simultaneous binding of Yersinia antigens and immunoglobulins was a prerequisite for the detection of ICs. The method will be valuable for research into the immunopathogenetic mechanisms leading to reactive arthritis after Yersinia infection.
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PMID:Detection of circulating Yersinia-immunoglobulin complexes by enzyme immunoassay (EIA). 351 76

Escherichia coli strains encoding the Yersinia pseudotuberculosis invasin protein are efficiently internalized by mammalian cells. Bacterial uptake into cultured cell lines became defective, however, if invasin was altered by fusion of its carboxyl terminus to E. coli alkaline phosphatase or by the addition of two hydrophobic amino acids to its carboxyl-terminal end. Probing with anti-invasin monoclonal antibodies revealed that the amino-terminal end of invasin was properly localized on the bacterial cell surface in strains encoding invasin with 2 additional amino acids, whereas the carboxyl terminus was not accessible to the monoclonal antibody. Therefore, the 2 additional amino acids interfered with the folding or orientation of the carboxyl terminus in the outer membrane. Alkylation experiments in the absence of reduction indicated that this defect was not caused by a gross inability to form a critical disulfide bond. Revertants were selected from a strain encoding this mutant protein by enriching for organisms able to enter cultured mammalian cells. The vast majority of revertants that were isolated following this enrichment contained a stop codon at the usual position found in the wild type inv gene. The most efficient of the remaining revertants resulted in the introduction of a glycine residue at the site of the wild type stop codon, presumably restoring proper conformation of the carboxyl-terminal region.
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PMID:Residues added to the carboxyl terminus of the Yersinia pseudotuberculosis invasin protein interfere with recognition by integrin receptors. 768 99

The iron starvation-induced, 2,042-amino-acid protein HMWP2 of Yersinia enterocolitica has two internal hydrophobic segments which might promote its export and association with the cytoplasmic membrane. To determine whether part of HMWP2 could be exported beyond the periplasmic face of the cytoplasmic membrane, we used TnphoA mutagenesis to construct 10 hybrid proteins in which periplasmic alkaline phosphatase (PhoA) was fused to the end of C-terminally truncated HMWP1 (at amino acid positions 1751 and 1753 two independent isolates]) had high alkaline phosphate activity (close to that of the native enzyme), both in Escherichia coli and in Y. pseudotuberculosis, indicating that the PhoA segment of the hybrid reached the periplasm. Deletion studies showed that the export signal resides in the second hydrophobic segment of HMWP2. This result would be compatible with the topology of the protein in the cytoplasmic membrane predicted from the distribution of charged amino acids at either end of the two hydrophobic segments. However, two hybrids in which the junction was even further toward the C terminus of HMMWP2 (at positions 1793 and 1999) had only weak alkaline phosphatase activity, suggesting that the predicted topology is incorrect. The location of HMWP2 was therefore determined by subcellular fractionation. The results indicate that HMPW2 is mainly cytoplasmic, consistent with its presumed role in the ATP-dependent, nonribosomal synthesis of an unknown peptide. We propose that the high alkaline phosphatase activity associated with some of the HMWP-2-PhoA hybrids results from the unmasking of the cryptic export signal activity in the second hydrophobic segment of HMPW2.
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PMID:Yersinia spp. HMWP2, a cytosolic protein with a cryptic internal signal sequence which can promote alkaline phosphatase export. 789 1

X-ray crystal structures of the Yersinia tyrosine phosphatase (PTPase) in complex with tungstate and nitrate have been solved to 2. 4-A resolution. Tetrahedral tungstate, WO42-, is a competitive inhibitor of the enzyme and is isosteric with the substrate and product of the catalyzed reaction. Planar nitrate, NO3-, is isosteric with the PO3 moiety of a phosphotransfer transition state. The crystal structures of the Yersinia PTPase with and without ligands, together with biochemical data, permit modeling of key steps along the reaction pathway. These energy-minimized models are consistent with a general acid-catalyzed, in-line displacement of the phosphate moiety to Cys403 on the enzyme, followed by attack by a nucleophilic water molecule to release orthophosphate. This nucleophilic water molecule is identified in the crystal structure of the nitrate complex. The active site structure of the PTPase is compared to alkaline phosphatase, which employs a similar phosphomonoester hydrolysis mechanism. Both enzymes must stabilize charges at the nucleophile, the PO3 moiety of the transition state, and the leaving group. Both an associative (bond formation preceding bond cleavage) and a dissociative (bond cleavage preceding bond formation) mechanism were modeled, but a dissociative-like mechanism is favored for steric and chemical reasons. Since nearly all of the 47 invariant or highly conserved residues of the PTPase domain are clustered at the active site, we suggest that the mechanism postulated for the Yersinia enzyme is applicable to all the PTPases.
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PMID:The X-ray crystal structures of Yersinia tyrosine phosphatase with bound tungstate and nitrate. Mechanistic implications. 870 35


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