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Biomedical subjects

B Auer

Publications and source records attributed to B Auer.

At least 37 records · Page 2Linked to original sources
PubMed1995

Regulation of the human poly(ADP-ribosyl) transferase promoter via alternative DNA racket structures.

Human nuclear poly(ADP-ribosyl) transferase (ADPRT) protein content in cells suggests that ADPRT expression is stringently controlled. Analysis of the 3 kb promoter sequence, which is required for high level expression, revealed an extraordinary architecture: several Sp1 motifs are located in the vicinity of the first exon but the closest CCAAT/TATA boxes are several hundred basepairs away. Four Alu type repetitive sequences are in the promoter structure. Within these Alu sequences there exist inverted repeat elements, which could form two mutually exclusive types of DNA tertiary structure consisting of quadruplex DNA and loops resembling rackets. Thereby, a CCAAT/TATA element would be moved to spatial vicinity of the Sp1 site activating the promoter. Deletion analysis showed the functional significance of these racket elements. We also obtained evidence for DNA racket structures when we studied mutational mechanisms in a human adenine phosphoribosyltransferase (APRT) deficient patient. One of his alleles harbours a novel complex type of deletion/insertion mutation. Based on several highly informative sequence features in this genomic region a model is proposed for the generation of this unusual type of mutation involving two steps: an initial targeting step and a subsequent complex rearrangement. This process includes the formation of a DNA racket structure, which resembles that of the ADPRT promoter. Thus we conclude that DNA racket structures seem to be of general importance in nature.

Base Sequence↗
PubMed1995

Pregnancy-related changes of carnitine and acylcarnitine concentrations of plasma and erythrocytes.

Total-, free-, and acylcarnitine concentrations were determined in whole blood, plasma, and red blood cells of 88 women during pregnancy. Already in the 12th week of gestation the mean whole blood carnitine level was significantly (p < 0.01) lower than those of the controls. From the 12th gestational week up to parturition there was a further significant (p < 0.01) decrease. This reduction of total carnitine in whole bloods was mainly caused by a significant (p < 0.01) decrease of free carnitine levels, since no marked changes of short chain acylcarnitine values were found throughout pregnancy. The contribution of red blood cell L-carnitine to whole blood carnitine increased significantly (p < 0.05) to 61% at delivery versus 39% (controls). In umbilical cord blood free and total carnitine levels were significantly (p < 0.05) higher than the corresponding maternal levels. The contribution of red blood cell L-carnitine to whole blood carnitine was higher in cord blood than in maternal blood. The results of the present study demonstrate that during pregnancy whole blood and plasma carnitine levels decrease to those levels found in patients with carnitine deficiency. Also the percentage of acylcarnitine on total carnitine, found in the present study, is characteristic for a secondary carnitine deficiency. Thus L-carnitine substitution in pregnant women, especially in risk pregnancies, may be advantageous.

Acylation↗
PubMed1994

The human ubiquitin-conjugating enzyme UbcH1 is involved in the repair of UV-damaged, alkylated and cross-linked DNA.

The human ubiquitin-conjugating enzyme UbcH1 shows 69% identity to the Saccharomyces cerevisiae RAD6/UBC2 which plays a key role in DNA repair. To examine the function of UbcH1 (formerly named E2, M(r) 17,000), [(1990) EMBO J. 9, 1431-1435]) we tested its ability to functionally substitute for yeast RAD6/UBC2 in the recovery of cells from various DNA damage. Complementation by expression of the human UbcH1 cDNA revealed that the UbcH1 carries out the function of S. cerevisiae RAD6/UBC2 in the repair of UV-damaged, alkylated and cross-linked DNA.

Alkylation↗
PubMed1994

Effect of transfection of human poly(ADP-ribose)polymerase in Chinese hamster cells on mutagen resistance.

Poly(ADP-ribose)polymerase (PARP) is a DNA-binding protein that is activated upon induction of DNA breaks and supposed to play a role in DNA repair. To elucidate the effect of overexpression of PARP on the resistance of cells to mutagens, Chinese hamster ovary cells (both the line CHO-9 and the mutagen-hypersensitive derivative 27-1) were transfected with the human PARP cDNA along with pSV2neo. Treatment of the transfected cell population with a high dose of MNNG and selection with G418 gave rise to a significant increase of neo+ clones, as compared to the control transfection with pSV2neo + salmon sperm DNA. The frequency of survivors in these mass culture experiments was lower, however, than after transfection with the bacterial ada gene encoding the DNA repair protein O6-alkylguanine-DNA alkyltransferase. Thus transfection of PARP cDNA in CHO cells is only weakly effective in inducing alkylation resistance. This was confirmed by analyzing the mutagen resistance of individual PARP transfectant clones derived from CHO-9 and 27-1 cells that expressed increased levels of PARP mRNA, protein and PARP activity. These strains were slightly more resistant to the toxic effect of MMS and showed a reduced frequency of MMS-induced chromosomal aberrations. CHO-9-PARP transfectants also gained resistance to UV. From these data we conclude that, in CHO cells, PARP is limiting in handling critical lesions during the repair process and that increase of the amount of PARP protein can elicit some protection against genotoxic effects of mutagens.

Animals↗
PubMed1994

Transcriptional regulation and autoregulation of the human gene for ADP-ribosyltransferase.

Human nuclear poly(ADP-ribosyl)transferase (ADPRT) modifies proteins with branched ADP-ribose-polymers. Various proteins, including ADPRT itself, serve as acceptors for polyADP-ribose. Target proteins include those controlling basic cellular processes such as DNA repair, differentiation and proliferation. Because of the outstanding features of this enzyme: automodification, several functional domains and central role in physiology of the cell, the molecular biology of ADPRT gained wide interest. The promoter structure contains several CCAAT/TATA boxes and SP1 sites. However, there is no CCAAT/TATA box in the neighbourhood of an SP1 site and, thus no obvious site for initiation of transcription. Within this region there are several noteworthy inverted repeats, which by internal basepairing could form two types of cruciform structures. Deletion analysis revealed that these cruciform structures have functional significance. Removal of one type increases the promoter activity, whereas removal of the other diminishes the promoter function. Overexpression of ADPRT from heterologous promoters (MMTV, SV40) leads to repression of the activity of the ADPRT promoter. Indeed, ADPRT was shown to bind specifically to one type of cruciform structure. This specific interaction indicates autorepression of the ADPRT gene: the enzyme ADPRT acts directly as a negative modulator of the activity of its own promoter.

Base Sequence↗
PubMed1994

The immunosuppressive substance 2-chloro-2-deoxyadenosine modulates lipoprotein metabolism in a murine macrophage cell line (P388 cells).

A recently developed immunosuppressive substance, 2-chloro-2-deoxyadenosine (2-CdA), was reported to inhibit monocyte functions at low concentration. Because macrophages play a key role in the formation of atherosclerotic plaques, it was of interest to study the effect of 2-CdA on cellular lipid metabolism. For this purpose we have used a macrophage cell line (P388) to perform incubation studies in the presence of acetylated low density lipoprotein (Ac-LDL) and 2-CdA. The addition of 2-CdA, in concentrations ranging from 5-20 nM, induced a dose-dependent decrease in cellular cholesterol content and in the amount of extracellular [14C]oleic acid (OA) incorporated into the cholesteryl ester (CE) fraction. The effect was maximized at 20 nM 2-CdA with an 86% reduction in cholesterol esterification compared to controls (P < 0.008). To evaluate the mechanism of interaction of 2-CdA with cellular lipid metabolism, deoxycytidine (dCyt) and 3-methoxybenzamide (3-MOB), substances known to antagonize the effect of 2-CdA in different ways, were co-administered with 2-CdA. dCyt, a competitive inhibitor of dCyt kinase, which catalyzes phosphorylation to the active metabolite, antagonized the effects of 20 nM 2-CdA, producing significantly greater incorporation of extracellular [14C]OA into the CE fraction than in the presence of 2-CdA alone (P < 0.0086). Co-incubation with 2-CdA and the poly-ADP-ribose synthetase inhibitor 3-MOB, which is known to render cells resistant to 2-CdA toxicity by preventing cellular nicotinamide adenine dinucleotide (NAD)- and adenosine triphosphase-depletion, also reversed the effect of 2-CdA on lipid accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed1993

Purification and characterization of NAD+:ADP-ribosyltransferase (polymerizing) from Dictyostelium discoideum.

A novel affinity-purification scheme based on the tight binding of NAD+:ADP-ribosyltransferase (polymerizing) [pADPRT; poly(ADP-ribose) polymerase; EC 2.4.2.30] to single-strand nicks in DNA, single-stranded patches and DNA ends has been developed to facilitate the purification of this enzyme from the lower eukaryote Dictyostelium discoideum. Two homogeneous forms of the enzyme, with M(r) values of 116,000 and 90,000, were prepared from D. discoideum by using poly(A) hybridized to oligo(dT)-cellulose as affinity material. The Km is 20 microM NAD+ for the 90,000-M(r) protein and 77 microM NAD+ for the 116,000-M(r) protein. The optimum conditions for the enzyme activity in vitro are 6-10 degrees C and pH 8. The time course is linear during the first 10 min of the reaction only. As in enzymes of higher eukaryotes, the activity is dependent on DNA and histone H1 and is inhibited by 3-methoxybenzamide, nicotinamide, theophylline, caffeine and thymidine.

Animals↗
PubMed1993

Genomic organization, localization, and allelic differences in the gene for the human neuropeptide Y Y1 receptor.

A 14-kilobase pair (kb) region of genomic DNA encoding the human neuropeptide Y Y1-receptor gene including 3'- and 5'-flanking sequences has been cloned and the human gene localized to chromosome 4q(31.3-32). In contrast to the contiguous structure of most G protein-coupled receptor genes, the NPY Y1 receptor gene is divided into three exons. A small 5'-exon of the mRNA untranslated region is separated by a 6-kb intron from the second exon. The coding region of the receptor is interrupted by a small intron, containing an in-frame stop codon, shortly after the proposed fifth transmembrane domain. In the 5'-flanking region a potential cAMP-response element and an AP-2 site, in addition to a TATA-like sequence and a typical CAAT, box are present. A single point mutation within the 6-kb intron generates a PstI polymorphic site with a highly informative allele frequency of 54:46% in the population.

Alleles↗
PubMed1993

Polyclonal antibody-based immunoradiometric assay for quantification of cholesteryl ester transfer protein.

Cholesteryl ester transfer protein (CETP) catalyzes the transfer of neutral lipids among plasma lipoproteins and in this way plays a prominent role in cholesterol metabolic routing and, thus, probably for atherosclerosis. Studies of this important protein in various clinical settings require the ability to accurately quantify CETP in plasma. In order to gain access to such a capability, an immunoradiometric assay (IRMA) for quantification of CETP was developed. CETP was purified from human plasma to apparent homogeneity and used for raising anti-CETP antibodies in rabbits. The specificity of the polyclonal antiserum obtained was demonstrated by inhibition assays and immunoblot analysis. Before use in the CETP-IRMA, the antibodies were affinity-purified by chromatography on CETP-Sepharose. Sensitivity of the CETP-IRMA was 0.1 ng, and intra- and interassay coefficients of variation were 2.9 and 8.0%, respectively. In 30 normolipidemic healthy subjects, the mean (+/- SD) CETP concentration was 1.1 (+/- 0.22) micrograms/ml of plasma; individual values ranged from 0.644 to 1.694 micrograms CETP/ml and agreed well with measurements of CETP activity of the same samples (r = 0.85).

Antibodies↗
PubMed1992

Inhibition of cell proliferation in Saccharomyces cerevisiae by expression of human NAD+ ADP-ribosyltransferase requires the DNA binding domain ("zinc fingers").

Constitutive expression of human nuclear NAD+: protein ADP-ribosyltransferase (polymerizing) [pADPRT; poly(ADP-ribose)polymerase; EC 2.4.2.30] as an active enzyme in Saccharomyces cerevisiae, under the control of the alcohol dehydrogenase promoter, was only possible with simultaneous inhibition of ADP-ribosylation by 3-methoxybenzamide. Induction of fully active pADPRT from the inducible galactose epimerase promoter resulted in inhibition of cell division and morphological changes reminiscent of cell cycle mutants. Expression of a pADPRT cDNA truncated at its 5' end had no influence on cell proliferation at all. Obviously the amino-terminal part of the DNA binding domain containing the first "zinc finger", which is essential for inducibility of pADPRT activity by DNA breaks, is also required for inhibition of cell growth on expression in yeast. Full-length as well as truncated pADPRT molecules were directed to the cell nucleus where the fully active enzyme produced large amounts of poly(ADP-ribose) by automodification. Since pADPRT turned out to be the only target for ADP-ribosylation in these cells, elevated levels of poly(ADP-ribose) were the most likely cause of inhibition of cell division, presumably resulting from interaction with chromosomal proteins.

Adenosine Diphosphate Ribose↗
PubMed1992

Fluorescence in situ mapping of the human nuclear NAD+ ADP-ribosyltransferase gene (ADPRT) and two secondary sites to human chromosomal bands 1q42, 13q34, and 14q24.

A 3.5-kb cDNA probe containing the 23 exons from the coding sequence of human nuclear NAD+ ADP-ribosyltransferase (poly [ADP-ribose] polymerase [ADPRT], E.C.2.4.2.30) was used to map the gene and two additional sites by nonisotopic in situ chromosomal hybridization. The previous localization of the structural gene on 1q42 was confirmed. Two other hybridization peaks on 13q34 and 14q24 suggested the presence of ADPRT pseudogenes.

Chromosome Mapping↗
PubMed1990

Primary structure of a DNA (N6-adenine)-methyltransferase from Escherichia coli virus T1. DNA sequence, genomic organization, and comparative analysis.

Escherichia coli virus T1 encodes a DNA (N6-adenine)-methyltransferase (M.T1) with the same sequence specificity as the E. coli DNA (N6-adenine)-methyltransferase (M.Eco dam). This enzyme was purified to homogeneity and a partial amino acid sequence determined. Oligonucleotides were constructed and used not only as probes to map the gene on the T1 genome, but also as primers in sequencing reactions to establish the nucleotide sequence of the M.T1 locus by primer extension. These data represent the first analysis of the genomic organization of bacterial virus T1 on a molecular level. Significant homology to E. coli consensus transcription and translation-initiation signals suggest that the gene for M.T1 is most probably under control of its own promoter. It may be transcribed as a polycistronic mRNA, together with a downstream open reading frame which codes for a polypeptide containing 83 amino acids (HP 83). Both the deduced primary and the secondary structure of the M.T1 were compared to those of other known DNA methyltransferases, especially those recognizing the sequence, GATC; there is little similarity of the T1 enzyme to the other members of this family.

Amino Acid Sequence↗
PubMed1990

Effects of haloperidol and clozapine on preprotachykinin-A messenger RNA, tachykinin tissue levels, release and neurokinin-1 receptors in the striato-nigral system.

The effects of haloperidol and clozapine on tachykinin tissue levels, preprotachykinin-A messenger RNA, spontaneous and potassium-evoked tachykinin release, dopamine D2 receptors, and [125I]Bolton-Hunter-substance P binding sites in the striato-nigral system were examined. Chronic administration (10 days) of the dopamine receptor antagonist haloperidol (2 mg/kg i.p.) significantly decreased tissue levels of substance P like-immunoreactivity and neurokinin A like-immunoreactivity in the striatum and the substantia nigra. The corresponding preprotachykinin-A mRNA was decreased in the striatum. Haloperidol did not affect the potassium-evoked tachykinin release in the substantia nigra but significantly increased the spontaneous release. Haloperidol increased the number of D2-receptors but left [125I]Bolton-Hunter-substance P binding sites, representing neurokinin 1 (NK-1) receptors, as determined by competition experiments with selective ligands, unchanged. Clozapine (30 mg/kg, i.m.) did not influence nigral and striatal tachykinin tissue levels, preprotachykinin-A mRNA and potassium-evoked release or spontaneous efflux in the substantia nigra, or D2-receptors and [125I]Bolton-Hunter-substance P binding sites. The present data indicate that neuroleptics influence the striato-nigral tachykinin system in different ways. Tachykinins may, therefore, contribute to the therapeutic and/or untoward effects of certain neuroleptic drugs.

Animals↗
PubMed1989

Human nuclear NAD+ ADP-ribosyltransferase: localization of the gene on chromosome 1q41-q42 and expression of an active human enzyme in Escherichia coli.

The gene for human nuclear NAD+ ADP-ribosyltransferase [NAD+:poly(adenosine diphosphate D-ribose) ADP-D-ribosetransferase, EC 2.4.2.30; pADPRT] was localized to chromosome 1 at q41-q42 by in situ hybridization with a pADPRT-specific cDNA probe. Expression of a pADPRT cDNA under control of the lac promoter in Escherichia coli induces the synthesis of a group of related proteins that were immunoreactive with pADPRT antibody and that had catalytic properties very similar to those of the human enzyme. Purification of this enzymatic activity was performed essentially as described for the human enzyme. The Km, pH optimum, optimal reaction temperature, and inhibition by 3-aminobenzamide and 3-methoxybenzamide were found to be similar for the recombinant and the human enzymes. The purified recombinant enzyme consists of two major proteins of Mr 99,000 and Mr 89,000. Both proteins show pADPRT activity in activity gel analysis with [32P]NAD+ as substrate. Microsequencing of these two proteins isolated by denaturing gel electrophoresis and deletion mutagenesis of the pADPRT expression plasmid shows that the Mr 99,000 and Mr 89,000 proteins derive from initiation of translation at internal translational start signals located within the pADPRT cDNA.

Amino Acid Sequence↗
PubMed1989

Human nuclear NAD+ ADP-ribosyltransferase(polymerizing): organization of the gene.

Human nuclear NAD+: protein ADP-ribosyltransferase(polymerizing) [pADPRT; poly(ADP-ribose)poly-merase; EC 2.4.2.30] is a DNA-dependent protein-modifying enzyme composed of several domains important for DNA binding, automodification, and NAD binding. We report that the human pADPRT gene is 43 kb in length and is split into 23 exons. All the intron-exon boundaries correspond to a canonical splice consensus sequence. Each of the four metal coordinating sites putatively forming the two zinc fingers of the DNA-binding domain is encoded separately. The automodification domain and the NAD-binding domain are coded for by 4 and 12 exons, respectively.

ADP Ribose Transferases↗