Biomedical subjects

H Cuypers

Publications and source records attributed to H Cuypers.

12 recordsLinked to original sources

PubMed2005

Multicenter evaluation of the NucliSens EasyQ HIV-1 v1.1 assay for the quantitative detection of HIV-1 RNA in plasma.

The Nuclisens EasyQ HIV-1 v1.1 assay (Biomerieux) is a real-time detection method combined with NASBA technology designed to measure plasma HIV-RNA. Its performance was assessed in 1008 clinical specimens collected from individuals infected with clade B (774) and non-B (234) HIV-1 variants at four European laboratories. The results were compared with those obtained using three other commercial viral load assays: Cobas Amplicor Monitor HIV-1 v1.5 (Roche), Versant HIV-1 RNA assay (Bayer) and Nuclisens HIV-1 QT (Biomerieux). Overall, the linearity, specificity and reproducibility of the EasyQ assay was comparable with that from the other tests. The correlation coefficient (R) between methodologies was 0.85 for Amplicor; 0.87 for Versant; and 0.91 for Nuclisens. The specificity of the assay was 99.4%. Of note, Versant missed 17% of specimens with non-B subtypes which could be detected by EasyQ, while Amplicor provided similar results than EasyQ. HIV-1 group O specimens were only detected by the EasyQ assay. In conclusion, the performance of the EasyQ assay seems to be similar to that of other HIV-1 viral load tests currently on the market, but it is more sensitive than Versant for HIV-1 non-B subtypes and shows a wider dynamic range than Amplicor. Moreover, as it incorporates the advantage of real-time detection procedures, it facilitates high throughput and short turnaround time.

France↗

PubMed1997

Studies on spontaneous promoter-up mutations in the transcriptional activator-encoding gene phIR and their effects on the degradation of phenol in Escherichia coli and Pseudomonas putida.

The activator-encoding gene phlR was identified upstream of the plasmid-encoded operon for phenol degradation in Pseudomonas putida strain H by cassette mutagenesis and DNA sequence analysis. The deduced amino acid sequence of PHLR shows high homology to DmpR of P. putida sp. CF600 and to the chromosomally encoded PhhR of P. putida P35X reported previously. Trans-activation of phenol degradation was observed when phlR was overexpressed in a phlR insertion mutant. Transconjugants of Escherichia coli carrying pPGH11, which contains the complete set of phl genes, are unable to grow on phenol as carbon source. However, two types of mutants were selected for further characterization that were able to metabolize phenol as sole source of carbon and energy. In both types of mutants enhanced expression of phlR is responsible for the Phl+ phenotype. In type I (pPGH13) a deletion of 1 bp made the -35 region and the spacing between the -35 and -10 regions of the phlR promoter more similar to the consensus structure. In type II (pPGH14) a duplication of the phlR 5' region was identified that includes part of the -35 motif and reduces the spacing between the -35 and -10 regions. In addition, due to the duplication of part of phlR, the distance from the phlR promoter to the catabolic phl operon is increased. Different transcriptional start sites have been identified by primer extension analysis in clones harboring pPGH14 or the wild type phlR. Quantitative primer extension analysis revealed that the greatest amount of phlR transcript is expressed from the partial, phlR duplication. Growth on phenol and phenol hydroxylase activity reflect the high level of phlR transcript in E. coli transconjugants. Overexpression of PhlR was also observed when pPGH14 was transferred into P. putida, and results in earlier induction of the phenol degradation operon relative to the wild-type strain.

Bacterial Proteins↗

PubMed1996

Carbon catabolite repression of phenol degradation in Pseudomonas putida is mediated by the inhibition of the activator protein PhlR.

Enzymes involved in (methyl)phenol degradation of Pseudomonas putida H are encoded by the catabolic operon (phlA-L) on plasmid pPGH1. Transcription of this operon by the sigma54 (RpoN)-containing RNA polymerase is positively controlled by the gene product of the divergently transcribed phlR in response to the availability of the respective substrate. Additionally, phenol degradation is subject to carbon catabolite repression induced by organic acids (e.g., succinate, lactate, and acetate) or carbohydrates (e.g., glucose and gluconate). Analysis of lacZ fusion to the catabolic promoter and quantified primer extension experiments indicate that carbon catabolite repression also occurs at the transcriptional level of the catabolic operon. In this study, it is furthermore shown that carbon catabolite repression is a negative control. Titration of the postulated negative controlling factor was exclusively observed when extra copies of functional phlR gene were present in the cell. We therefore conclude that PhlR is the target and that carbon catabolite repression of phenol degradation occurs by interfering with the activating function of PhlR.

Bacterial Proteins↗

PubMed1995

Multiple nosZ promoters and anaerobic expression of nos genes necessary for Pseudomonas stutzeri nitrous oxide reductase and assembly of its copper centers.

Respiration of N oxides (denitrification) by bacteria is expressed facultatively in response to environmental stimuli. We have studied the transcriptional organization of the nos gene cluster of Pseudomonas stutzeri. This cluster carries the information for a functional nitrous oxide reductase (NosZ) which catalyzes the last step of the denitrification process. The nos genes are transcribed in three units, nosR, nosZ, and nosDFY. Transcription of nosZ is initiated from six different promoters which extend over a region of about 200 bp. The activity of two promoters varies subject to different growth conditions. Promoter P3 is active preferentially under denitrifying conditions and presumably under the control of a homolog of the transcriptional regulator FNR. Promoter P2 is the most active start site under aerobiosis and likely to initiate the low constitutive expression of nosZ. Transcription of nosR, encoding a regulator for nosZ expression, and transcription of the nosDFY operon, required for the copper chromophore assembly of NosZ, are both initiated from a single promoter. Transcription of nosR and the nosDFY operon was shown by phoA and lacZ fusions to be activated under a lowered oxygen tension and the simultaneous presence of an N oxide. The enzymatic activities associated with the hybrid proteins suggest for NosR and NosF a location in the cytoplasmic membrane and the cytoplasm, respectively.

Base Sequence↗

PubMed1994

Nitric oxide reductase from Pseudomonas stutzeri. Primary structure and gene organization of a novel bacterial cytochrome bc complex.

Nitric oxide (NO) reductase is an integral membrane component of the anaerobic respiratory chain of Pseudomonas stutzeri that transforms nitrate to dinitrogen (denitrification). The enzyme catalyzes the reduction of NO to nitrous oxide. The structural genes for the NO reductase complex, norC and norB, were sequenced and their organization established by primer extension and Northern blot analysis. The norCB genes encoding the cytochrome c and cytochrome b subunits of the enzyme are contiguous and transcribed as a single 2.0-kb transcript. The promoter region has a canonical recognition motif for the transcriptional activator protein Fnr, centered at -40.5 nucleotides from the initiation site of transcription. No similarity of the derived gene products to known cytochromes of b- or c-type was found in a data bank search. Post-translational processing of the two subunits was limited to the removal of the terminal methionine to leave an N-terminal serine in either subunit. The mature cytochrome c subunit (16508Da, 145 residues) is predicted to be a bitopic protein with a single membrane anchor. The mature cytochrome b subunit (53006Da, 473 residues) is a putatively polytopic, strongly hydrophobic membrane-bound protein with 12 potential transmembrane segments. Several histidine and proline residues were identified with potentially structural and/or functional importance. Mutational inactivation of NO reductase by deletion of norB or the norCB genes affected strongly the in vivo activity of respiratory nitrite reductase (cytochrome cd1), but to a much lesser extent the expression level of this enzyme. In turn, mutational inactivation of the structural gene for cytochrome cd1, nirS, or loss of in vivo nitrite reduction by mutation of the nirT gene, encoding a presumed tetraheme cytochrome, lowered the expression level of NO reductase to 5-20%, but hardly its catalytic activity. The cellular concentration of NO reductase increased again on restoration of nitrite reduction in the nirS::Tn5 mutant MK202 by complementation with nirS or with the heterologous nirK gene, encoding the Cu-containing nitrite reductase from Pseudomonas aureofaciens. Thus, NO may be required as an inducer for its own reductase. Our results show that the nitrite-reducing system and the NO-reducing system are not operating independently from each other but are interlaced by activity modulation and regulation of enzyme synthesis.

Amino Acid Sequence↗

PubMed1993

Anaerobic control of denitrification in Pseudomonas stutzeri escapes mutagenesis of an fnr-like gene.

The synthesis of proteins necessary for the respiratory reduction of nitrate to dinitrogen is induced in most denitrifying bacteria by a shift to anaerobiosis. A homolog of the fur gene, which encodes a redox-active transcriptional activator in Escherichia coli, was isolated from Pseudomonas stutzeri by using the anr gene of Pseudomonas aeruginosa as the hybridization probe (R. G. Sawers, Mol. Microbiol. 5:1469-1481, 1991). The coding region was located on a 3-kb SmaI fragment. An open reading frame of 735 nucleotides, designated fnrA, had the coding potential for a protein of 244 amino acids (M(r) = 27,089) with 51.2% positional identity to the Fnr protein of E. coli and 86.1% to the Anr protein of P. aeruginosa. The fnrA gene gave a single transcript of 0.85 kb and complemented nitrate-dependent anaerobic growth of an fnr deletion mutant of E. coli. An open reading frame immediately downstream of fnrA encoded adenine phosphoribosyltransferase (EC 2.4.2.7). Mutations in fnrA were generated in vitro by insertional mutagenesis followed by gene replacement. Gene inactivation was shown by loss of the fnrA transcript and detection of an arginine deiminase (EC 3.5.3.6)-negative phenotype in the mutants. However, neither the enzymatic activities nor the levels of anaerobic expression of the respiratory enzymes nitrate reductase (EC 1.7.99.4), nitrate reductase (EC 1.9.3.2), NO reductase (EC 1.7.99.7), and N2O reductase (EC 1.7.99.6) were changed in fnrA mutants versus the P. stutzeri wild type. A promoter-probe vector for Fnr-dependent transcription was activated anaerobically in the fnrA mutants, suggesting the existence of a second Fnr homolog in the same bacterium. The Fnr-binding motifs, apparent in the promoter region of genes encoding denitrification components of P. stutzeri, are likely to be recognized by this second Fnr homolog. Preliminary evidence indicates also the presence of the catabolite activator protein, Crp, in P. stutzeri.

Amino Acid Sequence↗

PubMed1992

Derived amino acid sequences of the nosZ gene (respiratory N2O reductase) from Alcaligenes eutrophus, Pseudomonas aeruginosa and Pseudomonas stutzeri reveal potential copper-binding residues. Implications for the CuA site of N2O reductase and cytochrome-c oxidase.

The nosZ genes encoding the multicopper enzyme nitrous oxide reductase of Alcaligenes eutrophus H16 and the type strain of Pseudomonas aeruginosa were cloned and sequenced for structural comparison of their gene products with the homologous product of the nosZ gene from Pseudomonas stutzeri [Viebrock, A. & Zumft, W. G. (1988) J. Bacteriol. 170, 4658-4668] and the subunit II of cytochrome-c oxidase (COII). Both types of enzymes possess the CuA binding site. The nosZ genes were identified in cosmid libraries by hybridization with an internal 1.22-kb PstI fragment (NS220) of nosZ from P. stutzeri. The derived amino acid sequences indicate unprocessed gene products of 70084 Da (A. eutrophus) and 70695 Da (P. aeruginosa). The N-terminal sequences of the NosZ proteins have the characteristics of signal peptides for transport. A homologous domain, extending over at least 50 residues, is shared among the three derived NosZ sequences and the CuA binding region of 32 COII sequences. Only three out of nine cysteine residues of the NosZ protein (P. stutzeri) are invariant. Cys618 and Cys622 are assigned to a binuclear center, A, which is thought to represent the CuA site of NosZ and is located close to the C terminus. Two conserved histidines, one methionine, one aspartate, one valine and two aromatic residues are also part of the CuA consensus sequence, which is the domain homologous between the two enzymes. The CuA consensus sequence, however, lacks four strictly conserved residues present in all COII sequences. Cys165 is likely to be a ligand of a second binuclear center, Z, for which we assume mainly histidine coordination. Of 23 histidine residues in NosZ (P. stutzeri), 14 are invariant, 7 of which are in regions with a degree of conservation well above the 50% positional identity between the Alcaligenes and Pseudomonas sequences. Conserved tryptophan residues are located close to several potential copper ligands. Trp615 may contribute to the observed quenching of fluorescence when the CuA site is occupied.

Alcaligenes↗

PubMed1992

NosR, a membrane-bound regulatory component necessary for expression of nitrous oxide reductase in denitrifying Pseudomonas stutzeri.

The regulatory element NosR was identified within the nos region of the denitrification gene cluster of Pseudomonas stutzeri ZoBell (ATCC 14405) and characterized. It is essential for expression of the N2O reductase encoded by nosZ immediately downstream of nosR. The nosR region was initially identified by Tn5 mutagenesis (W. G. Zumft, K. Döhler, and H. Körner, J. Bacteriol. 163:918-924, 1985). It consists of a single open reading frame of 2,172 nucleotides and has the coding capacity for an 81.9-kDa protein. The codon usage for nosR, with its high G + C content of 62.4 mol% and a preference for G or C at the third position, is characteristic for a Pseudomonas gene. Hydropathy analysis classified NosR as an integral membrane protein with at least seven membrane-spanning segments. No similarity to known bacterial regulator proteins was found in a data bank search. However, the C terminus of NosR shows sequence similarity to the cysteine clusters of several 2[4Fe-4S] bacterial ferrodoxins. A monocistronic mRNA for nosZ which allowed us to monitor NosR function was identified. Complementation of Nos- mutant MK418 (nosR::Tn5) with the nosR gene supplied in trans restored nosZ transcription and expression of a catalytically active N2O reductase. In addition to evidence of the requirement for NosR, indirect evidence for involvement of the transcriptional regulator Fnr is presented.

Amino Acid Sequence↗

PubMed1991

Risk factors in hepatitis C virus-infected blood donors.

Risk factors of parenteral and nonparenteral exposure to hepatitis C virus (HCV) infection were studied in 125 blood donors in The Netherlands who were positive for anti-HCV on enzyme-linked immunosorbent assay (ELISA). Risk factors were related to confirmatory test results of four-antigen recombinant immunoblot assay (4-RIBA) and polymerase chain reaction (PCR) of the HCV 5' untranslated region. Twelve (10%) of the 125 anti-HCV C100 ELISA-positive blood donors were 4-RIBA positive. Eleven (92%) of 12 4-RIBA-positive blood donors were PCR positive, and all 113 remaining 4-RIBA-negative or -indeterminate donors were PCR negative. Eleven (92%) of 12 4-RIBA-positive blood donors had a risk factor of parenteral exposure, as compared to 17 (15%) of 113 4-RIBA-negative or -indeterminate donors. The prevalence of confirmed HCV infection among Amsterdam blood donors is calculated at 0.04 percent; parenteral exposure appears to be the major risk factor for HCV infection.

Blood Donors↗

PubMed1988

The defective En-I102 element encodes a product reducing the mutability of the En/Spm transposable element system of Zea mays.

Genetic and molecular analysis has revealed a specific En-element of deletion derivative (En-I102) which reduces En/Spm-induced mutability. In the presence of En-I102 the excision frequency of both the autonomous En-1 element and the inhibitor element Spm-I5719A is reduced and excision occurs later in development. The 3697 bp long En-I102 element is derived from En-1 by an internal deletion of 4590 bp removing nucleotides 1862-6451. The promoter at the left end and sequences required for polyadenylation are retained in En-I102. It is transcribed to yield predominantly a 1.8 kb poly(A) RNA. cDNA analysis of this transcript indicated that it contains the coding capacity for a 386 amino acid polypeptide. This polypeptide shares homology with En/Spm encoded functions and we suggest that it interferes with transposition at the protein level.

Alleles↗

PubMed1986

Molecular analysis of the En/Spm transposable element system of Zea mays.

The nucleotide sequence of the autonomous transposable element En-1 isolated from the wx-844::En-1 allele has been determined. En-1 is 8287 bp long. The structure of the mosaic gene 1, coding for the major En transcript, has been established. The promoter gene 1 is located in the highly structured left end of the element and the gene spans almost the entire length of En-1. The first intron of gene 1 is 4434 nucleotides long and contains two large open reading frames, 2714 bp and 761 bp in size, which hybridize to minor RNA species in Northern blot experiments.

Alleles↗

PubMed1985

Plant transposable elements generate the DNA sequence diversity needed in evolution.

Two germinal and 16 somatic reversion events induced by the Enhancer (En) transposable element system at the wx-8::Spm-I8 allele of Zea mays were cloned and studied by sequence analysis. Excision of the Spm-I8 receptor element from the wx gene results in various mutant DNA sequences. This leads to altered gene products, some of which are still capable of restoring the wild-type phenotype. Possible 'foot-print' sequences that may have arisen by the excision of transposable elements were observed when intron sequences of the wild-type (wx+) and mutant (wx-m8) alleles of the wx gene were compared. The sequence divergence generated by visitation of a locus by plant transposable elements is discussed with respect to the molecular evolution of the new gene functions.

Base Sequence↗