Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0348321 (Haemophilus)
 15,372 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An enzyme-linked immunosorbent assay (ELISA) is described for detection of anti-capsular antibodies against Haemophilus influenzae type b. Polyribosephosphate was covalently bonded to poly-L-lysine before adsorption to microtiter plates. ELISA immunoglobulin G and immunoglobulin M anti-polyribosephosphate antibody titers were comparable to total anti-polyribosephosphate antibody concentration determined by radioimmunoassay. The ELISA technique will be useful for further investigations of host response to infections due to H. influenzae type b but is not intended to be used as a serological method for documenting H. influenzae type b infections.
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PMID:Enzyme-linked immunosorbent assay for detection of capsular antibodies against Haemophilus influenzae type b: comparison with radioimmunoassay. 660 75

Forty-nine bacterial strains representing five species known to interact with human plasminogen were tested for the ability to bind the two major human plasminogen activators, t-PA and urokinase. The bacterial species tested included Haemophilus influenzae, Neisseria meningitidis, Streptococcus pyogenes, Streptococcus equisimilis and human group G streptococci. All N. meningitidis and 11 of 14 H. influenzae strains displayed substantial binding of t-PA with values in the range of 20-46%. On the contrary, none of the streptococcal strains bound significant amounts of tPA. With urokinase no binding could be found for any of the bacterial species tested. Scatchard analysis with a selected H. influenzae strain (HI23354) demonstrated 10,000 receptors per bacterium for t-PA with a Kd value of about 20 nmol l-1. The corresponding values with a selected N. meningitidis strain (Mo 52) was 8500 receptors per bacterium and 70 nmol l-1. t-PA binding could be reduced about 40% by the addition of 10 mmol l-1 of the lysine analogue epsilon-aminocaproic acd (EACA) whereas no inhibitory effect could be demonstrated with arginine. Addition of 2 mumol l-1 of plasminogen which is enough to occupy all bacterial sites for plasminogen did not interfere with the t-PA binding, suggesting that the receptors for t-PA and plasminogen are distinct. Using very high plasminogen concentrations however, t-PA binding could be reduced by about 50% possibly due to an interaction between t-PA and plasminogen in the fluid phase. Our results demonstrate the occurrence of a previously unknown type of bacterial receptor that is capable of specifically binding t-PA.
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PMID:Binding of tissue-type plasminogen activator (t-PA) to Neisseria meningitidis and Haemophilus influenzae. 780 68

The nucleotide sequences of the quinolone resistance-determining regions of the gyrA and parC genes from five ciprofloxacin-resistant strains of Haemophilus influenzae (MICs, 2 to 32 micrograms/ml) isolated from patients with cystic fibrosis and three ciprofloxacin-susceptible strains of H. influenzae (MICs, < or = 0.1 micrograms/ml) were determined. Four of the five resistant strains possessed at least one amino acid substitution in each of the GyrA and ParC fragments studied. The mutations identified in GyrA were a serine at residue 84 (Ser-84) to Leu or Tyr and Asp-88 to Asn or Tyr. ParC mutations were in positions exactly analogous to those identified in GyrA, namely, Ser-84 to Ile and Glu-88 to Lys. The Glu-88 to Lys ParC substitution was identified only in high-level ciprofloxacin-resistant strains. These mutations have been shown to be the origin of the observed resistance after transformation into ciprofloxacin-susceptible H. influenzae isolates. These results suggest that H. influenzae isolates require at least one amino acid substitution in both GyrA and ParC in order to attain significant levels of resistance to quinolones.
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PMID:Ciprofloxacin-resistant Haemophilus influenzae strains possess mutations in analogous positions of GyrA and ParC. 880 76

asd mutants of Gram-negative and some Gram-positive bacteria have an obligate requirement for diaminopimelic acid (DAP), an essential constituent of the cell wall of these organisms. In environments deprived of DAP, for example mammalian tissues, they will undergo lysis. This was previously exploited to develop vaccine strains of Salmonella typhimurium and cloning vectors containing asd as an in vivo selectable marker. As a first step for development of such systems for Pseudomonas aeruginosa, the asd gene from wild-type strain PAO1 was cloned by a combined approach of PCR amplification from chromosomal DNA, construction of mini-libraries and by complementation of an Escherichia coli delta asd mutant. The nucleotide sequence of a 2433 bp Smal-Nsil fragment was determined. This fragment contained the C-terminal 47 nucleotides of leuB, encoding 3-isopropylmalate dehydrogenase; asd, encoding aspartate-beta-semialdehyde dehydrogenase (Asd); and orfA, whose product showed similarity to the Asd proteins from Vibrio spp. By subcloning, asd was localized to a 1.24 kb DNA fragment which in an E. coli T7 expression system strongly expressed a 40,000 Da protein. The amino acid sequence was deduced from the DNA sequence. A comparison of the Asd proteins from P. aeruginosa, E. coli and Haemophilus influenzae revealed greater than 63% identity, demonstrating the conserved nature of Asd in Gram-negative bacteria, and defined the active-site-containing consensus sequence GGNCTVXMLMXXXLGLF as a possible signature motif. Chromosomal delta asd mutants were isolated. They were auxotrophic for DAP, lysine, methionine and threonine, and lysed in the absence of DAP. Genetic analyses indicated that orfA probably is naturally frame-shifted and does not contribute to the Asd phenotype. By PFGE, the asd gene was mapped to between coordinates 1.89 and 2.15 Mbp, or 37-40 min, on the 5.9 Mbp P. aeruginosa chromosome.
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PMID:Molecular genetic analysis of the region containing the essential Pseudomonas aeruginosa asd gene encoding aspartate-beta-semialdehyde dehydrogenase. 908 74

Hydrolysis of N-succinyl-L,L-diaminopimelic acid by the dapE-encoded desuccinylase is required for the bacterial synthesis of lysine and meso-diaminopimelic acid. We have investigated the catalytic mechanism of the recombinant enzyme from Haemophilus influenzae. The desuccinylase was overexpressed in Escherichia coli and purified to homogeneity. Steady-state kinetic experiments verified that the enzyme is metal-dependent, with a Km for N-succinyl-L,L-diaminopimelic acid of 1.3 mM and a turnover number of 200 s-1 in the presence of zinc. The maximal velocity was independent of pH above 7 but decreased with a slope of 1 below pH 7. The pH dependence of V/K was bell-shaped with apparent pKs of 6.5 and 8.3. Both L,L- and D,L-diaminopimelic acid were competitive inhibitors of the substrate, but d,d-diaminopimelic acid was not. Solvent kinetic isotope effect studies yielded inverse isotope effects, with values for D2OV/K of 0.62 and D2OV of 0.78. Determination of metal stoichiometry by ICP-AES indicated one tightly bound metal ion, while sequence homologies suggest the presence of two metal binding sites. On the basis of these observations, we propose a chemical mechanism for this metalloenzyme, which has a number of important structurally defined homologues.
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PMID:Hydrolysis of N-succinyl-L,L-diaminopimelic acid by the Haemophilus influenzae dapE-encoded desuccinylase: metal activation, solvent isotope effects, and kinetic mechanism. 967 18

Selenocysteine synthase from Escherichia coli is a pyridoxal-5'-phosphate-containing enzyme which catalyses the conversion of seryl-tRNA(Sec) into selenocysteyl-tRNA(Sec). Analysis of amino acid sequences indicated that selenocysteine synthase belongs to the alpha/gamma superfamily of pyridoxal-5'-phosphate-dependent enzymes. To identify the lysine residue carrying the prosthetic group, the genes coding for the selenocysteine synthases from Moorella thermoacetica and Desulfomicrobium baculatum were cloned and sequenced and their derived amino acid sequences were aligned with those from E. coli and Haemophilus influenzae. Three lysine residues were found to be conserved; they were mutated into asparagine and one of them, Lys295, was found to be essential for activity. Proteolytic fragmentation of the E. coli enzyme reduced with borohydride, and mass-spectrometric and sequence analysis of the chromophoric peptide proved that Lys295 was modified. Kinetic analysis of the enzyme showed that thiophosphate served as a substrate leading to cysteyl-tRNA(Sec) synthesis, albeit with a 330-fold lower catalytic efficiency. Selenide and, to a much lesser degree, sulfide could also be used by the enzyme but only at much higher concentrations. These data together with the finding that selenophosphate synthetase is highly specific for selenide indicate that the phosphate moiety of selenophosphate provides selenocysteine synthase with the discrimination specificity against sulfur.
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PMID:Bacterial selenocysteine synthase--structural and functional properties. 968 79

The gene (denoted aroQp.pheA) encoding the bifunctional P-protein (chorismate mutase-P/prephenate dehydratase) from Xanthomonas campestris was cloned. aroQp.pheA is essential for L-phenylalanine biosynthesis. DNA sequencing of the smallest subclone capable of functional complementation of an Escherichia coli phenylalanine auxotroph revealed a putative open reading frame (ORF) of 1200 bp that would encode a 43,438-Da protein. AroQp.PheA exhibited 51% amino acid identity with a Pseudomonas stutzeri homologoue and greater than 30% identities with AroQp.PheA proteins from Haemophilus influenzae, Neisseria gonorrhoeae, and a number of enteric bacteria. AroQp.PheA from X. campestris, when expressed in E. coli, possesses a 40-residue amino-terminal extension that is lysine-rich and that is absent in all of the AroQp.PheA homologues known at present. About 95% of AroQp.PheA was particulate and readily sedimented by low-speed centrifugation. Soluble preparations of cloned AroQp.PheA exhibited a native molecular mass of 81,000 Da, indicating that the active enzyme species is a homodimer. These preparations were unstable after purification of about 40-fold, even in the presence of glycerol, which was an effective protectant before fractionation. When AroQp.PheA was overproduced by a T7 translation vector, unusual inclusion bodies having a macromolecular structure consisting of protein fibrils were observed by electron microscopy. Insoluble protein collected at low-speed centrifugation possessed high catalytic activity. The single band obtained via SDS-PAGE was used to confirm the translational start via N-terminal amino acid sequencing. A perspective on the evolutionary relationships of monofunctional AroQ and PheA proteins and the AroQp.PheA family of proteins is presented. A serC gene located immediately upstream of X. campestris aroQp.pheA appears to reflect a conserved gene organization, and both may belong to a single transcriptional unit.
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PMID:The aroQ and pheA domains of the bifunctional P-protein from Xanthomonas campestris in a context of genomic comparison. 968 22

Analysis of the proteome of Haemophilus influenzae by two-dimensional polyacrylamide gel electrophoresis on conventional Tris-glycine gels does not usually result in efficient separation of the proteins in the 5-20 kDa range, which are mainly accumulated in the lower acidic and basic regions. In order to improve the separation of the low molecular mass proteins, we used homogeneous Tricine gels of two urea concentrations in the second-dimensional separation. The Tricine gel systems allowed the efficient and reproducible separation of the proteins of the microorganism with masses between 5 and 20 kDa, however, no proteins with masses below 5 kDa could be visualized. Approximately 80 proteins migrating in the 5-25 kDa region were identified by matrix assisted laser desorption/ionization - mass spectrometry, of which 40 identified for the first time. The digestion of the low mass proteins often produced only few peptides, which were insufficient for confident identification by mass spectrometry. Therefore, the identification was occasionally achieved by a sequential digestion with two proteases, trypsin or endoproteinase Lys-C as first and carboxypeptidase P as second enzyme. The gel system described may be useful for the efficient separation of low molecular mass proteins from other organisms to construct standard maps.
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PMID:Reference map of the low molecular mass proteins of Haemophilus influenzae. 971 65

Abs using the kappaII-A2 V gene segment predominate the human Ab repertoire to the Haemophilus influenzae b (Hib) polysaccharide (PS). All A2 anti-Hib PS Abs sequenced to date possess a 10-amino acid L chain complementarity-determining region-3 (CDR-3) having an insertional arginine (Arg) at position 95a, the V-J junction. These findings suggest an essential requirement for this conserved Arg residue in determining Hib PS-binding affinity. We examined this requirement by performing chain recombination experiments in which a series of A2 L chains, differing at position 95a, were combined individually with an Fd region known to generate a Hib PS-combining site when paired with an A2-Arg(95a)-Jkappa1 V region. Hib PS binding of the recombinant Fabs was evaluated quantitatively using a radioantigen-binding assay. Fabs having A2 L chains with either Arg or lysine in position 95a in combination with Jkappa1 gave equivalent and strongest binding to Hib PS. Fabs having A2-Jkappa1 L chains with either tyrosine, glycine, alanine, leucine, serine, or threonine in position 95a, or having an A2-Arg(95a)-Jkappa3 L chain, gave intermediate binding. Fabs having A2-Jkappa1 L chains with glutamate or aspartate at 95a or with no junctional residue showed little or no Hib PS binding. These results demonstrate the importance of L chain junctional residue, as well as Jkappa usage and CDR-3 length, in determining Hib PS-binding affinity. Contrary to expectation, an Arg junctional residue is not essential for generating either high or intermediate affinity-binding sites.
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PMID:Role of kappa II-A2 light chain CDR-3 junctional residues in human antibody binding to the Haemophilus influenzae type b polysaccharide. 975 4

Structural aspects of human TCRs that allow the activation of autoreactive T cells by diverse microbial peptides were examined using two human myelin basic protein (MBP)-specific T cell clones. The TCR sequences of these clones differed only in the N region of TCR-alpha and -beta since the clones had the same Valpha-Jalpha and Vbeta-Jbeta rearrangements. The two clones had a similar fine specificity for the MBP peptide, except for the P5 position of the peptide (lysine). In the crystal structure of the HLA-DR2/MBP peptide complex, P5 lysine is a prominent, solvent-exposed residue in the center of the DR2/MBP peptide surface. Five microbial peptides with conservative or nonconservative changes at the P5 position (lysine to arginine, serine, or proline) activated one of these clones. In contrast, the other clone was activated only by three of these peptides which had a conservative lysine to arginine change at P5. The degree of specificity/degeneracy in recognition of the P5 side chain was the key difference between these TCRs since the Escherichia coli/Haemophilus influenzae peptide stimulated both clones when the P5 position was substituted from serine to arginine. These results demonstrate that the complementarity-determining region 3 loops contribute to the degree of degeneracy in peptide recognition by human MBP-specific TCRs.
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PMID:Structural features of autoreactive TCR that determine the degree of degeneracy in peptide recognition. 988 4


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