PubMed Health⌕ Search

Biomedical subjects

A Böck

Publications and source records attributed to A Böck.

At least 73 records · Page 4Linked to original sources
PubMed1995

Characterisation of a protease from Escherichia coli involved in hydrogenase maturation.

The large subunits of nickel-containing hydrogenases are synthesised in a precursor form which, after nickel incorporation, is processed by proteolytic cleavage at the C-terminal end. The protease involved in processing of HycE, the large subunit of hydrogenase 3 from Escherichia coli, was purified by three chromatographic steps to apparent homogeneity. Its gene was identified by using a hybridisation probe generated by PCR with oligonucleotide primers the sequence of which was derived from the N-terminal and internal amino acid sequences. Determination of the nucleotide sequence showed that the gene is located distally and as a hitherto uncharacterised gene within the hyc operon, coding for hydrogenase 3 components. It was designated hycI. The HycI protease has a molecular mass of 17 kDa and is a monomer. Its cleavage reaction is not inhibited by conventional inhibitors of serine and metalloproteases, which correlates with the fact that the sequence does not contain signature motifs characteristic of serine-, metallo-, cysteine- or acid proteases. Homologous genes are present in other transcriptional units coding for hydrogenases.

Amino Acid Sequence↗
PubMed1995

tRNA genes and pathogenicity islands: influence on virulence and metabolic properties of uropathogenic Escherichia coli.

The uropathogenic Escherichia coli strain 536 (O6:K15:H31) carries two unstable DNA regions on its chromosome which were termed pathogenicity islands (Pais). Both pathogenicity islands, Pai I and Pai II, are incorporated into tRNA specific loci: Pai I is located in the tRNA gene for selenocysteine (selC), and Pai II is integrated in the leucine-specific tRNA locus leuX. Mutant strain 536-21 has lost the two pathogenicity islands together with the intact tRNA genes. While 536 is a virulent strain, 536-21 has lost a number of properties, including in vivo virulence. In previous publications we reported that the genes coding for two haemolysins (hly I, hly II) and P-related fimbria (prf) are located on the Pais. In this paper, we demonstrate that the expression of other gene products influencing metabolic properties in addition to in vivo virulence are strongly dependent on the intact tRNA loci selC and leuX. In order to determine the influence of the two tRNAs on the expression of these properties, the genes selC and leuX were cloned from the genome of strain 536 and then introduced into the mutant 536-21. Our results clearly show that the seleno-cysteine-specific tRNA (tRNA(Sec)) directly influences the ability of the bacteria to grow under anaerobic conditions, because selenocysteine is part of the enzyme formate dehydrogenase (FDH) which is involved in mixed acid fermentation. The rare leucine-specific tRNA5(Leu), encoded by leuX, influences a number if properties including type 1 fimbria production, flagellation and motility, production of enterobactin and serum resistance, and is also necessary for full in vivo virulence. While the tRNA(Sec) is directly involved in the production of FDHs, the leuX specific tRNA5(Leu) appears to influence the expression of various factors through specific transcriptional or translational control mechanisms.

Anaerobiosis↗
PubMed1995

Effector-mediated stimulation of ATPase activity by the sigma 54-dependent transcriptional activator FHLA from Escherichia coli.

The FHLA protein is the transcriptional regulator of the genes of the formate regulon from Escherichia coli. The protein shares homology with the sigma 54-dependent regulators of the NTRC family in the central and C-terminal domains but differs in possessing an extended N terminus lacking the aspartate residue which is the site of phosphorylation. Purified FHLA displays intrinsic ATPase activity which is stimulated weakly by formate and DNA. The presence of both formate and DNA carrying the upstream regulatory sequence to which FHLA binds leads to a large increase in the rate of ATP hydrolysis. Hypophosphite, a structural analog of formate, and azide, a transition state analog of formate, also stimulate ATPase activity, supporting the conclusion that formate is a direct ligand of FHLA. Half-maximal saturation of FHLA with formate took place at around 5 mM, and half-maximal saturation with target DNA took place at around 50 nM. The stimulation of ATPase activity by formate was conferred by a decrease in the apparent Km for ATP, whereas the effect of the DNA binding site also affected the Kcat of the reaction. The other nucleoside triphosphates, GTP, UTP, and CTP, competed with ATP cleavage by FHLA, suggesting at least their binding to FHLA. The specific ATPase activity of FHLA was dependent on the concentration of FHLA in the assay, especially in the presence of DNA and formate. Direct liganding of the effector, therefore, leads to the same consequence as phosphorylation for the NTRC-type regulators, namely, stimulation of ATPase activity.

Adenosine Triphosphatases↗
PubMed1995

Identification of the transcriptional activator controlling the butanediol fermentation pathway in Klebsiella terrigena.

The gene budR, whose product is responsible for induction of the butanediol formation pathway under fermentative growth conditions in Klebsiella terrigena, has been cloned and sequenced. This gene is separated from the budABC operon by a nontranslated region of 106 bp and transcribed in the opposite direction. budR codes for a protein of molecular weight 32,124, the sequence of which exhibits characteristics of regulators belonging to the LysR family. When transferred into the heterologous host Escherichia coli, budR activates expression of budA'-lacZ transcriptional and translational fusions with a regulatory pattern identical to that in K. terrigena, namely, induction by acetate, low pH, and anaerobiosis. Induction by acetate was specific, indicating that it is the physiological inducer. Primer extension analysis located the start site of transcription to two positions, 23 and 24 bp upstream of the budR initiation codon, and also showed that BudR strongly autoregulates its own expression. The products of fhlA, arcA, hip, ntrA, and katF did not influence expression of the bud operon. A mutation in fnr, however, led to a threefold increase in expression, indicating that Fnr acts as a repressor. The results support the notion that BudR coordinates the activity of the energy-conserving, nonreductive, but acidifying acetate formation pathway with the expression of the non-energy-conserving, reductive, but nonacidifying butanediol pathway.

Amino Acid Sequence↗
PubMed1994

Purification and DNA-binding properties of FHLA, the transcriptional activator of the formate hydrogenlyase system from Escherichia coli.

FHLA is the transcriptional activator for the expression of the genes coding for components of the formate hydrogenlyase system of Escherichia coli. The cloned fhlA gene was overexpressed under selected growth conditions, and a purification protocol for the FHLA protein was developed. Purified FHLA in the native state is a homotetramer. It binds to the upstream regulatory sequences of the fdhF gene and of the intergenic region between the divergently transcribed hyc and hyp operons. An additional binding site of FHLA located between the hycA and hycB genes was identified. While binding to the hypA-hycA intergenic region is responsible for activation of expression of the hyc operon, the newly identified site is required for the activation of the sigma 54-dependent promoter located upstream of the hyp operon. Formate seems to have no effect on the DNA-binding properties of FHLA. The binding sites of FHLA were characterized by DNase I footprinting; sequence motifs putatively involved in the interaction with FHLA are described.

Base Sequence↗
PubMed1994

Regulated expression in vitro of genes coding for formate hydrogenlyase components of Escherichia coli.

Purified FHLA, the transcriptional activator of the formate regulon from Escherichia coli, is able to efficiently stimulate transcription from the sigma 54-dependent promoters of the fdhF, hyp, and hyc transcriptional units. Expression was dependent on the presence of sigma 54, of the upstream activatory sequence (UAS), and of formate. Hypophosphite, a formate analogue, could substitute for formate in vitro suggesting that formate per se was active in regulation. The integration host factor (IHF) had a direct effect on the expression (in vivo and in vitro) of the hyp and hyc genes but not of the fdhF gene. Binding of IHF within the region between the hyp and the hyc operon could be shown. A model is proposed for the transcriptional regulation of the inversely oriented hyp and hyc operons. It involves two upstream regulatory sequences, one between the hyp and the hyc operon (IR1), and the other between hycA and hycB (IR2). The UAS situated within IR1 is responsible for activation of the hyc operon, that within IR2 for activation of the hyp operon. A supramolecular transcription complex is proposed which involves the binding of IHF to a site located between the UAS and the promoter responsible for transcription of the hyc operon.

Bacterial Proteins↗
PubMed1994

Eukaryotic selenocysteine inserting tRNA species support selenoprotein synthesis in Escherichia coli.

Although the tRNA species directing selenocysteine insertion in prokaryotes differ greatly in their primary structure from that of their eukaryotic homologues they share very similar three-dimensional structures. To analyse whether this conservation of the overall shape of the molecules reflects a conservation of their functional interactions it was tested whether the selenocysteine inserting tRNA species from Homo sapiens supports selenoprotein synthesis in E. coli. It was found that the expression of the human tRNA(Sec) gene in E.coli can complement a lesion in the tRNA(Sec) gene of this organism. Transcripts of the Homo sapiens and Xenopus laevis tRNA(Sec) genes synthesised in vitro were amino-acylated by the E.coli seryl-tRNA ligase although at a very low rate and the resulting seryl-tRNA(Sec) was bound to and converted into selenocysteyl-tRNA(Sec) by the selenocysteine synthase of this organism. Selenocysteyl-tRNA(Sec) from both eukaryotes was able to form a complex with translation factor SELB from E.coli. Although the mechanism of selenocysteine incorporation into seleno-proteins appears to be rather different in E.coli and in vertebrates, we observe here a surprising conservation of functions over an enormous evolutionary distance.

Animals↗
PubMed1994

The formation of diselenide bridges in proteins by incorporation of selenocysteine residues: biosynthesis and characterization of (Se)2-thioredoxin.

A system was devised which allows the efficient substitution of cysteine residues in a protein by selenocysteine. It involves overexpression of the respective gene with the aid of the T7 promotor/polymerase system in a cysteine auxotrophic strain. The induction of the T7 polymerase formation was performed in cysteine-supplemented medium followed by wash-out of the cysteine and production of the desired gene product in the presence of selenocysteine. The system was applied to substitute the two cysteine residues in Escherichia coli thioredoxin. Analysis of the purified gene product by electrospray mass spectrometry and HPLC revealed that both cysteine residues were replaced in approximately 75-80% of the protein, only one cysteine residue was substituted in about 5-10%, and no substitution had taken place in 12-17% of the protein. The occurrence of diselenide, seleno-sulfur, and disulfide bridges in the purified gene product was revealed by ES/MS and chemical modification studies. The diselenide bridge represents an entity in protein structures which has hitherto not been described. The redox property of the selenocysteine variant of thioredoxin [(Se)2-thioredoxin] was found to be substantially different from that of thioredoxin. Only the latter could be reduced under native conditions in the presence of an excess of beta-mercaptoethanol. The oxidized (Se)2-thioredoxin was then separated from the selectively reduced and carboxymethylated protein by anion-exchange chromatography. The purity of the isolated (Se)2-thioredoxin was at least 92%.

Chromatography, High Pressure Liquid↗
PubMed1994

Maturation of the large subunit (HYCE) of Escherichia coli hydrogenase 3 requires nickel incorporation followed by C-terminal processing at Arg537.

Purification of the large subunit, HYCE, of Escherichia coli hydrogenase 3 revealed that it is a nickel-containing polypeptide, which is subject to C-terminal proteolytic processing. This processing reaction could be performed in vitro with partially purified components, yielding a low-molecular mass C-terminal peptide which was resolved in a Tricine/SDS/polyacrylamide gel. N-terminal sequencing of this peptide revealed that proteolytic cleavage occurred at the C-terminal side of the arginine residue at position 537, which corresponds to the histidine residue in the highly conserved motif, DPCXXCXXH, of other (NiFe) hydrogenases thought to be involved in active site nickel coordination. Nickel-containing HYCE precursor for in vitro processing, was partially purified from strain HD708 (delta hycH) in the presence of the reducing agent dithiothreitol. Using 2-mercaptoethanol instead of dithiothreitol provided pure precursor, which was, however, no longer susceptible to in vitro processing; it proved to be devoid of nickel indicating that nickel incorporation into the HYCE precursor is a prerequisite for processing. This conclusion was supported by the finding that HYCE precursor from strain HD708 (delta hycH) chromatographed with radioactivity from 83Ni incorporated in vivo and could be processed in vitro, whereas HYCE precursor from strain BEF314 (delta hypB-E) lacking the nickel insertion system appeared to be devoid of nickel and was not sensitive to in vitro processing.

Amino Acid Sequence↗
PubMed1994

Recognition of the mRNA selenocysteine insertion sequence by the specialized translational elongation factor SELB.

In Escherichia coli the unusual amino acid selenocysteine is incorporated cotranslationally at an in-frame UGA codon. Incorporation of selenocysteine relies, in part, on the interaction between a specialized elongation factor, the SELB protein, and a cis-acting element within the mRNA. Boundary and toeprint experiments illustrate that the SELB-GTP-Sec-tRNA(Sec) ternary complex binds to the selenoprotein encoding mRNAs fdhF and fdnG, serving to increase the concentration of SELB and Sec-tRNA(Sec) on these mRNAs in vivo. Moreover, toeprint experiments indicate that SELB recognizes the ribosome-bound message and that, upon binding, SELB may protrude out of the ribosomal-mRNA track so as to approach the large ribosomal subunit. The results place the mRNA-bound SELB-GTP-Sec-tRNA(Sec) ternary complex at the selenocysteine codon (as expected) and suggest a mechanism to explain the specificity of selenocysteine insertion. Cis-acting mRNA regulatory elements can tether protein factors to the translation complex during protein synthesis.

Bacterial Proteins↗
PubMed1994

Tonsillectomy and the immune system: a long-term follow up comparison between tonsillectomized and non-tonsillectomized children.

Immunological functions of the tonsils and possible effects of their removal are still controversial. One reason for this is the lack of long-term follow-up investigations after tonsillectomy. In the present study selected parameters of the cellular and humoral immune systems of 160 children 0.5-11 years after tonsillectomy (mean 6.6 +/- 2.1 years) were compared to those of 302 age-matched non-tonsillectomized children. In tonsillectomized children the incidence of infections of the upper respiratory tract was not increased compared to the non-tonsillectomized control group. Slightly increased percentages of CD 21 + cells, raised counts of CD4+ cells, absolute and relative increases in DR+ cells and a raised CD4+ DR count was found mainly in tonsillectomized boys, while lymphocyte subpopulations of tonsillectomized girls remained unaffected. Tonsillectomized children had lower IgA levels, but the complement system was not altered in either sex. These findings show that while tonsillectomy may lead to certain changes in the cellular and humoral immune systems, these alterations are clinically insignificant and no increased frequency of immunomodulated diseases should be expected.

Adolescent↗
PubMed1994

Factors contributing to the occurrence and predictability of bronchial hyperresponsiveness to methacholine in children.

Using a stepwise logistic regression analysis, we investigated clinical data, allergologic findings, spirometric data, and the cellular and humoral immune system in order to gain new insights into the role these parameters play in bronchial hyperresponsiveness to methacholine in children and to create a model for the prediction thereof. Bronchial hyperresponsiveness, which was found in 124 of 462 children (26.8%), was observed to have been influenced by an increased level of eosinophils, the positivity of the skin prick test for any of the allergens tested, a decreased baseline forced expiratory volume in 1 second (FEV1) (percent predicted), a decreased maximum expiratory flow at 50% expiration as a percent of forced vital capacity, and a decreased level of kappa-chain-assembled immunoglobulins. Logit analysis disclosed that the influence of all other parameters on the occurrence of bronchial hyperresponsiveness was of no further statistical significance. The degree of bronchial hyperresponsiveness (provocative dose causing a 20% fall in FEV1) showed a statistically significant correlation with the eosinophil count (Spearman's r = -0.198) and FEV1 (percent predicted) (Spearman's r= 0.203). Our findings suggest that allergic sensitization and eosinophilic reaction in children are major factors in contributing to the occurrence of bronchial hyperresponsiveness to methacholine.

Allergens↗
PubMed1994

Reticulocyte maturity pattern analysis as a predictive marker of erythropoiesis in paediatrics. Part I: Evaluation of age-dependent reference values.

Reticulocyte quantification in peripheral blood samples is a commonly used diagnostic indicator of erythropoietic activity. A methodology based on flow cytometry additionally separates reticulocytes into three groups by fluorescence staining of the residual RNA. This identifies cells as high (HFR), medium (MFR) and low (LFR) fluorescence intensity reticulocytes. In the present study an automated counter was evaluated and tested for its clinical applicability in paediatrics. In part I, reference intervals for different periods of childhood were determined. Except for the neonatal period there was no age-dependence so that children aged one week to 16 years have been summarized in one group. The wide variations we found in preterm children can be explained by different erythropoietic stimuli as the result of anaemia in infants with very low birthweight. No significant differences could be found between the sexes, not even at the onset of puberty. When using the reticulocyte maturity pattern analysis in clinical practice, the data give a helpful indication of the efficiency of the erythropoietic system.

Adolescent↗
PubMed1994

Reticulocyte maturity pattern analysis as a predictive marker of erythropoiesis in paediatrics. Part II: Pilot study for clinical application.

Reticulocyte quantification in peripheral blood samples is a commonly used diagnostic indicator of erythropoietic activity. A methodology based on flow cytometry additionally separates reticulocytes into 3 groups by fluorescence staining of the residual RNA. This identifies cells as 'high (HFR), medium (MFR) and low (LFR) fluorescence intensity' reticulocytes. In part II of the study we looked for the clinical applicability in paediatrics. Selected groups of patients with ineffective erythropoiesis, i.e. suffering from renal failure, oncologic patients with suppressed bone marrow activity caused by chemotherapy and anaemic new-born infants have been observed longitudinally for their reticulocyte maturity profiles. Data were compared to the commonly used parameters RBC, Hb and Hct. In all cases in which effective erythropoiesis returned documented by a normalization of standard blood parameters, HFR cells reacted significantly earlier than the traditional markers. These preliminary observations suggest the reticulocyte maturity pattern analysis can be used as an additional aid in diagnosis and as a helpful parameter for the monitoring of any anaemic situation.

Anemia↗
PubMed1994

Genes coding for the selenocysteine-inserting tRNA species from Desulfomicrobium baculatum and Clostridium thermoaceticum: structural and evolutionary implications.

The genes (selC) coding for the selenocysteine-inserting tRNA species (tRNA(Sec)) from Clostridium thermoaceticum and Desulfomicrobium baculatum were cloned and sequenced. Although they differ in numerous positions from the sequence of the Escherichia coli selC gene, they were able to complement the selC lesion of an E. coli mutant and to promote selenoprotein formation in the heterologous host. The tRNA(Sec) species from both organisms possess all of the unique primary, secondary, and tertiary structural features exhibited by E. coli tRNA(Sec) (C. Baron, E. Westhof, A. Böck, and R. Giegé, J. Mol. Biol. 231:274-292, 1993). The structural and functional properties of the tRNA(Sec) species from prokaryotes analyzed thus far support the notion that tRNA(Sec) may be an evolutionarily conserved structure whose function in the primordial genetic code was to decode UGA with selenocysteine.

Base Sequence↗
PubMed1993

Solution structure of selenocysteine-inserting tRNA(Sec) from Escherichia coli. Comparison with canonical tRNA(Ser).

Selenocysteine-inserting tRNAs (or tRNA(Sec)) are structurally untypical tRNAs that are charged by seryl-tRNA synthetase before being recognized by the selenocysteine synthase that converts serine into selenocysteine. tRNA(Sec) from Escherichia coli contains 95 nucleotides and is the longest tRNA known to date, in contrast to canonical tRNA(Ser), 88 nucleotides-long. We have studied its solution conformation by chemical and enzymatic probing. Global structural features were obtained by cobra venom and S1 nuclease mapping, as well as by probing with Pb2+. Accessibilities of phosphate groups were measured by ethylnitrosourea probing. Information about positions in bases involved in Watson-Crick pairing, in stacking or in tertiary interactions were obtained by chemical probing with dimethylsulfate, diethylpyrocarbonate, kethoxal and carbodiimide. On the basis of these chemical data, a three-dimensional model was constructed by computer modeling and compared to that of canonical tRNA(Ser). tRNA(Sec) resembles tRNA(Ser) at the level of its T-arm and anticodon-arm conformations, as well as at the joining of the D- and T-loops by a tertiary Watson-Crick G19-C56 interaction. Its extra-long variable arm is a double-stranded structure closed by a four nucleotide loop that is linked to the body of the tRNA in a way different from that found in tRNA(Ser). As anticipated from the peculiar features of the sequence in the D-loop and at the junction of amino acid and D-arms, tRNA(Sec) possesses a novel but restricted set of tertiary interactions in the core of its three-dimensional structure: a G8-A21-U14 triple pair and a novel interaction between C16 of the D-loop and C59 of the T-loop. A third triple interaction involving C15-G20a-G48 is suggested but some experimental evidence for it is still lacking. It is furthermore concluded that the D-arm has six base-pairs instead of three, as in canonical class II tRNA(Ser), with the D-loop containing only four nucleotides. Finally, the amino acid accepting arm forms a stack of eight Watson-Crick base-pairs (instead of 7 in other tRNAs). The biological relevance of this model with regard to interaction with seryl-tRNA synthetase and enzymes from the selenocysteine metabolism is discussed.

Adenine↗