Biomedical subjects

D Häring

Publications and source records attributed to D Häring.

15 recordsLinked to original sources

PubMed2001

Combined occurrence of a heterozygous missense mutation in the protein C gene and allelic exclusion of one protein S allele leading to severe venous thrombosis.

Individuals with more than one defect in the natural anticoagulant system exhibit an increased risk for thrombosis. We report on a family with two cases of combined protein C (PROC) and protein S (PROS) deficiency, five cases of isolated PROC deficiency Type I, and two cases of isolated PROS deficiency Type I. PROC and PROS deficiency were documented by functional and immunologic tests. The sequencing of all exons and splice junctions of the PROC gene led to the identification of a new, unpublished G-->A transition at nt 8490, leading to an exchange of alanine 259 by threonine. The mutation was present in all family members with PROC deficiency. The carriers of the isolated PROC mutation were asymptomatic at ages of 4, 7, 10, 11, and 80 years. The combination of the PROC mutation with a PROS deficiency in two family members triggered venous thromboembolism at age 31 and 6 years, respectively. The PROS deficiency was associated with complete exclusion of one PROS allele. Two family members with isolated PROS deficiency are still asymptomatic at age 21 and 9 years, respectively. Our findings in this family suggest that the heterozygous mutation at codon 259 of the PROC gene represents a mild thrombotic risk factor and only confers a high thrombotic risk in combination with a second defect, such as the complete exclusion of one PROS allele.

Adult↗

PubMed2001

No isochores in the human chromosomes 21 and 22?

The human genome is described in the literature as being composed of the isochores, i.e., long (hundreds of kilobases) segments with a homogeneous (G + C) content. We calculated the (G + C) content variations along the DNA molecules of the human chromosomes 21 and 22 and found the variations to be higher everywhere compared to the randomized sequences. Hence the (G + C) content is certainly not homogeneous on the isochore scale in the two human chromosomes. In addition, we found no significant difference between the two human molecules and the genome of E. coli regarding the (G + C) content variations. Hence no isochores are either present in the DNA molecules of the human chromosomes 21 and 22, or the isochores are also present in the genome of Escherichia coli. In any case, the present communication demonstrates that the isochores should be defined in unambiguous molecular terms if they are to be used for an up-to-date genome structure characterization.

Algorithms↗

PubMed2001

Specific host-guest interactions in a protein-based artificial transaminase.

Artificial enzymes can be created by covalent attachment of a catalytic active group to a protein scaffold. Recently, we assembled an artificial transaminase by conjugation of intestinal fatty acid binding protein (IFABP) with a pyridoxamine derivative via a disulfide bond; the resulting construct catalyzed a transamination reaction 200-fold faster than free pyridoxamine. To identify the origin of this increased catalytic efficiency computer modeling was first used to identify two putative residues, Y14 and R126, that were in close proximity to the gamma-carboxylate group of the substrate, alpha-ketoglutartate. These positions were mutated to phenylalanine and methionine, respectively, and used to prepare semisynthetic transaminases by conjugation to pyridoxamine (Px) or an N-methylated derivative (MPx). Kinetic analysis of the resulting constructs showed that the R126M mutation reduced substrate affinity 3- to 6-fold while the additional Y14F mutation had a negligible effect. These results are consistent with a model for substrate recognition that involves an electrostatic interaction between the cationic guanidinium group of R126 and the anionic carboxylate from the substrate. Interestingly, one of the conjugates that contains an N-methylated pyridoxamine catalyzes a transamination reaction with a k(cat)' value of 1.1h(-1) which is the fastest value for k(cat) we have thus far obtained and is 34-fold greater than that for the free cofactor in the absence of the protein.

Amino Acid Substitution↗

PubMed2001

Mosaic structure of the DNA molecules of the human chromosomes 21 and 22.

We calculated nucleotide distribution curves along the DNA molecules of the human chromosomes 21 and 22, their correlations in more than 10,000 equidistant positions, and subjected the correlations to cluster analysis. The cluster analysis demonstrated that both DNA molecules were composed of two types of segments exhibiting qualitatively different correlations. The segments differed most in the correlation of the distribution curves of cytosine and guanine, which was very high in type I segments but weak in type II segments. The type I and II segments also significantly differed in the correlations of the distribution curves of adenine with thymine. In addition, adenine strongly anticorrelated with cytosine but this anticorrelation was uniform along both chromosomes and, therefore, it did not contribute to the distinction of the two types of segments. The segments were up to 100 kbp long but they had nothing in common with isochores. Building blocks of the mosaic structure of the DNA molecules of the human chromosomes 21 and 22 are very similar but different in several interesting aspects from those of E. coli.

Chromosomes, Human, Pair 21↗

PubMed2000

Synthesis of a cationic pyridoxamine conjugation reagent and application to the mechanistic analysis of an artificial transaminase.

An N-methylated, cationic pyridoxamine conjugation reagent was synthesized and tethered via a disulfide bond to a cysteine residue inside the cavity of intestinal fatty acid binding protein. The conjugate was characterized and the kinetic parameters compared to its nonmethylated pyridoxamine analogue. Kinetic isotope effects were used for further mechanistic analysis. Taken together, these experiments suggest that a step distinct from deprotonation of the ketimine in the pyridoxamine to pyridoxal reaction is what limits the rate of the artificial transaminase IFABP-Px. However, the internal energetics of reactions catalyzed by the conjugate containing the N-methylated cofactor appear to be different suggesting that the MPx reagent will be useful in future experiments designed to alter the catalytic properties of semisynthetic transaminases.

Carrier Proteins↗

PubMed2000

Escherichia coli genome is composed of two distinct types of nucleotide sequences.

We calculated correlations of the nucleotide distributions along the E. coli genome. Subsequent cluster analysis of the correlation distributions showed that the genome was composed of two qualitatively different types of nucleotide sequences. The first type exhibited strong correlations of the genomic distributions of A with T and G with C, and high anticorrelations of A with C and G with T. In contrast, the second type was characterized by weak or negligible correlations typical of randomized sequences. Both types of sequences were almost equally abundant in the E. coli genome and their length varied from several hundred nucleotides to about 70 kilobases. They were not disjunct with respect to their (G + C) content but the high correlations and anticorrelations were rather characteristic for (A + T)-rich genomic segments. We offer possible explanations of the mosaic structure of the E. coli genome.

Base Composition↗

PubMed2000

The 4G/4G genotype at nucleotide position -675 in the promotor region of the plasminogen activator inhibitor 1 (PAI-1) gene is less frequent in young patients with minor stroke than in controls.

Genetic risk factors play an important role in the aetiology of vascular diseases. The insertion/deletion polymorphism (4G/5G) in the promotor region of the plasminogen activator inhibitor 1 (PAI-1) gene has been associated with an increased risk of myocardial infarction. We investigated 136 patients with minor stroke (MS) and transient ischaemic attack (TIA) and found a prevalence of 0.32 for the 4G/4G genotype in patients compared with 0.42 in 115 age-matched healthy controls. The 4G/4G genotype was significantly less frequent among 61 patients symptomatic before the age of 60 years (prevalence 0.20) than in 75 patients symptomatic after 60 years of age (prevalence 0.42; odds ratio). Our results indicate that the 4G/4G genotype is not a risk factor for MS or TIA and may even be protective in young patients.

Case-Control Studies↗

PubMed1999

Variations of the mononucleotide and short oligonucleotide distributions in the genomes of various organisms.

We calculated the variation coefficients of the mononucleotide and short oligonucleotide distributions in over 1700 long genomic sequences originating from six organisms to demonstrate that the human and Escherichia coli genomic sequences were the least and the most uniform, respectively. The most non-random genomic distributions were exhibited by the four canonical nucleotides, followed by the strong and weak nucleotides, while the distributions of purine or pyrimidine nucleotides and especially the distributions of (A+C) and (G+T) were significantly more uniform even in the human genome. In the human and mouse genomes, the highest coefficients of variation were further observed with the oligonucleotides where CG was combined with the strong nucleotides while its combination with the weak nucleotides significantly decreased the variation which, however, was still very high. High variation was also exhibited by the remaining oligonucleotides composed exclusively of the strong nucleotides or those containing only weak nucleotides. On the other hand, the distributions of oligonucleotides containing similar and especially the same numbers of the strong and weak nucleotides, but no CG or TA dinucleotide, were the most uniform. The information following from the present analysis will be useful not only in the identification of important genomic regions but also in computer simulations of the genomic nucleotide sequences in order to trace and reproduce the pathways of genome evolution.

Animals↗

PubMed1999

Biotransformations with peroxidases.

Enzymes are chiral catalysts and are able to produce optically active molecules from prochiral or racemic substrates by catalytic asymmetric induction. One of the major challenges in organic synthesis is the development of environmentally acceptable chemical processes for the preparation of enantiomerically pure compounds, which are of increasing importance as pharmaceuticals and agrochemicals. Enzymes meet this challenge! For example, a variety of peroxidases effectively catalyze numerous selective oxidations of electron-rich substrates, which include the hydroxylation of arenes, the oxyfunctionalizations of phenols and aromatic amines, the epoxidation and halogenation of olefins, the oxygenation of heteroatoms and the enantioselective reduction of racemic hydroperoxides. In this review, we summarize the important advances achieved in the last few years on peroxidase-catalyzed transformations, with major emphasis on preparative applications.

Biotransformation↗

PubMed1999

Chemical engineering of enzymes: altered catalytic activity, predictable selectivity and exceptional stability of the semisynthetic peroxidase seleno-subtilisin.

The increasing demand for enzymes as highly selective, mild, and environmentally benign catalysts is often limited by the lack of an enzyme with the desired catalytic activity or substrate selectivity and by their instability in biotechnological processes. The previous answers to these problems comprised genetically engineered enzymes and several classes of enzyme mimics. Here we describe the potential of chemical enzyme engineering: native enzymes can be modified by merely chemical means and basic equipment yielding so-called semisynthetic enzymes. Thus, the high substrate selectivity of the enzymatic peptide framework is combined with the catalytic versatility of a synthetic active site. We illustrate the potential of chemically engineered enzymes with the conception of the semisynthetic peroxidase seleno-subtilisin. First, the serine endoprotease subtilisin was crystallized and cross-linked with glutaraldehyde to give cross-linked enzyme crystals which were found to be insoluble in water or organic solvents and highly stable. Second, serine 221 in the active site (Enz-OH) was chemically converted into an oxidized derivative of selenocystein (Enz-SeO2H). As a consequence, the former proteolytic enzyme gained peroxidase activity and catalyzed the selective reduction of hydroperoxides. Due to the identical binding sites of the semisynthetic peroxidase and the protease, the substrate selectivity of seleno-subtilisin was predictable in view of the well-known selectivity of subtilisin.

Binding Sites↗

PubMed1999

Cross-linked enzyme crystals.

The active site of cross-linked enzyme crystals can be further modified by chemical means, yielding a new type of chemically engineered enzyme. In the past year, the characteristics of cross-linked protein crystals as microporous materials have been studied and used as stationary phases in chromatographic separations.

Biotransformation↗

PubMed1999

Correlations and anticorrelations among nucleotide distributions along the genomes of various organisms.

We have analyzed correlations of nucleotide distributions along more than 50 megabases of the longest sequenced parts of the human, mouse, Drosophila, Arabidopsis, yeast, E.coli and three kinds of viral genomes. The strongest correlations were observed between the distributions of C and G, in particular in the genome of Drosophila. This correlation was much weaker, though still strong, in the human genome and E.coli that exhibited the same level of this correlation. The C/G correlation hardly originates from the isochores because the isochores were not reported to occur in the genomes of Drosophila and E. coil. The genomic distribution curves of adenine and thymine were also positively correlated in all analyzed organisms except for the yeast where they were anticorrelated. Still stronger anticorrelations were, however, observed between the genomic distributions of A and C and between G and T. These genomic distributions anticorrelated almost generally and very strong. These anticorrelations are likely to originate from point mutations resulting from unrepaired GA mispairing as a replication intermediate. The C/A or G/T anticorrelation or compensation is a very strong and general new phenomenon that shapes the genomic nucleotide sequences.

Animals↗

PubMed1998

Reasoning enantioselectivity and kinetics of seleno-subtilisin from the subtilisin template.

The active-site serine (Ser221) of subtilisin Carlsberg(from Bacillus licheniformis) and subtilisin BPN' (fromBacillus amyloliquefaciens) was chemically converted into a selenocystein. Contrary to subtilisin's protease activity the semisynthetic seleno-subtilisin catalyzed the reduction of hydroperoxides. Enantioselectivity and kinetics of this reaction were studied by kinetic resolution of five racemic alkyl aryl hydroperoxides catalyzed by the seleno-subtilisin variants. Due to the identical tertiary structure of subtilisin and seleno-subtilisin, the enzymes have comparable substrate binding properties. Thus, a rational screening for suitable peroxidase substrates featuring structural characteristics of known subtilisin substrates was enabled. The enantioselective recognition of (S)-configured alkyl aryl hydroperoxides by seleno-subtilisin was comprehensible by subtilisin's preference for comparable (S)-alkyl aryl amines or alcohols. The analysis of chiral products by multidimensional gas chromatography revealed enantiomeric excesses up to 98%. Kinetics of seleno-subtilisin were rationalized on the basis of the established substrate-catalyst interactions of the subtilisin framework. The Carlsberg and BPN' peroxidase variants revealed typical differences in turnover numbers (kcat) and Michaelis-Menten affinity constants (Km) already known from subtilisin variants. Turnover numbers of seleno-subtilisin BPN' were lower and Km values were higher in comparison to Carlsberg variant. Substrate affinity of several substituted 1-arylethyl hydroperoxides to seleno-subtilisin was reasonable in comparison to corresponding aryl boronic acid inhibitors of subtilisin.

Bacillus↗

PubMed1997

Analysis of heterocyclic aromatic amines in wine by high-performance liquid chromatography-electrospray tandem mass spectrometry.

High-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS-MS) was used for the analysis of heterocyclic aromatic amines (HAAs) in 25 wines from various regions. In 24 wines under study HAAs were detected in the low ng/l range. 2-Amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alpha C) were found to be the most widely distributed HAAs in the wines under study. There was a difference in the presence of HAAs in white and red wines. In the latter, higher concentrations were found, with MeA alpha C in amount ranging from < 7.5 to 107 ng/l. Compared to the quantities of HAAs evaluated in meat and meat products, the amounts found in wines are 100-1000-fold lower.

Amines↗

PubMedDate not supplied

Enzymes by design: chemogenetic assembly of transamination active sites containing lysine residues for covalent catalysis.

Artificial enzymes can be created by covalent conjugation of a catalytic active group to a protein scaffold. Here, two transamination catalysts were designed via computer modeling and assembled by chemically conjugating a pyridoxamine moiety within the large cavity of intestinal fatty acid binding protein. Each catalyst included a lysine residue, introduced via site-directed mutagenesis, that promotes catalysis by covalent interactions with the pyridoxamine group. Evidence for such interactions include the formation of a Schiff base with the pyridoxal form of the catalyst and a rate versus pH dependence that is bell shaped; both of these features are manifested in natural transaminases. The resulting constructs operate with high enantioselectivity (83-94% ee) and increase the rate of reaction as much as 4200-fold over the rate in the absence of the protein; this is a modest (12-fold) increase in catalytic efficiency (kcat/KM) compared to the conjugate lacking the lysine residue. Most importantly, these artificial aminotransferases are the first examples of designed bioconjugates capable of covalent catalysis, highlighting the potential of this chemogenetic approach.

Carrier Proteins↗