PubMed Health⌕ Search

Biomedical subjects

A H Andersen

Publications and source records attributed to A H Andersen.

At least 37 records · Page 2Linked to original sources
PubMed1998

Characterization of DNA topoisomerase II alpha/beta heterodimers in HeLa cells.

In mammalian cells, DNA topoisomerase II is the product of two distinct genes encoding the alpha and beta isoforms of the enzyme. Besides homodimeric topoisomerase IIalpha and IIbeta, we have recently shown that alpha/beta heterodimers constitute a third population of topoisomerase II in HeLa cells. We found that topoisomerase II heterodimers are not restricted to HeLa cells but exist in different mammalian cell types, and up to 25% of the total topoisomerase IIbeta population is involved in heterodimer formation. Studies of topoisomerase II phosphorylation in HeLa cells show that heterodimers are phosphorylated in vivo to a significantly lower level compared to homodimeric alpha enzymes, but in contrast to the latter neither heterodimers nor topoisomerase IIbeta homodimers coprecipitate together with a kinase activity that is able to mediate their phosphorylation. However, both enzymes can still be phosphorylated by exogenously added casein kinase II. The differential phosphorylation of topoisomerase II heterodimers suggests an alternative regulation of this topoisomerase II subclass compared to the homodimeric topoisomerase IIalpha counterparts.

3T3 Cells↗
PubMed1996

Human DNA topoisomerases II alpha and II beta can functionally substitute for yeast TOP2 in chromosome segregation and recombination.

The ability of the human DNA topoisomerase II alpha and II beta isozymes to complement functional defects conferred by conditional top2 mutations in Saccharomyces cerevisiae has been investigated. At the restrictive temperature, top2 strains show multiple abnormalities, including an inability to complete mitotic and meiotic division owing to a defect in chromosome segregation, and hyper-recombination within the repetitive rDNA gene cluster. We show that the human topoisomerases II alpha and II beta can each support both vegetative growth and the production of viable spores in a top2-4 mutant at the restrictive temperature. Similarly, both human isozymes can rescue a strain carrying a top2 gene disruption, and suppress hyper-recombination within the rDNA gene cluster. We conclude that the human topoisomerase II alpha and II beta isozymes are functionally interchangeable with yeast topoisomerase II and suggest that any isozyme-specific roles in human cells are likely to be dependent upon factors other than inherent differences in catalytic ability between the alpha and beta isozymes.

Antigens, Neoplasm↗
PubMed1996

Active heterodimers are formed from human DNA topoisomerase II alpha and II beta isoforms.

DNA topoisomerase II is a nuclear enzyme essential for chromosome dynamics and DNA metabolism. In mammalian cells, two genetically and biochemically distinct topoisomerase II forms exist, which are designated topoisomerase II alpha and topoisomerase II beta. In our studies of human topoisomerase II, we have found that a substantial fraction of the enzyme exists as alpha/beta heterodimers in HeLa cells. The ability to form heterodimers was verified when human topoisomerases II alpha and II beta were coexpressed in yeast and investigated in a dimerization assay. Analysis of purified heterodimers shows that these enzymes maintain topoisomerase II specific catalytic activities. The natural existence of an active heterodimeric subclass of topoisomerase II merits attention whenever topoisomerases II alpha and II beta function, localization, and cell cycle regulation are investigated.

Amino Acid Sequence↗
PubMed1996

Cortical activation in confrontation naming.

Functional magnetic resonance imaging was used to detect cortical activation in the right and left perisylvian cortex of seven young adult right-handed volunteers in response to a letter fluency task and to a visual naming task using standardized line drawings. Both letter fluency and visual naming activated left dorsolateral prefrontal cortex (Brodmann's areas 6, 9, 44 and 45). Only visual naming activated area 37 (a cortical region with strong connections to visual association areas), visual association area 19, and areas 39 and 21 previously shown to activate with auditory semantic tasks. This study supports a role for area 37 as participant in a visual lexicosemantic processing network which may otherwise overlap the auditorysemantic network.

Adult↗
PubMed1996

Mapping drug-induced changes in cerebral R2* by Multiple Gradient Recalled Echo functional MRI.

A multiple Gradient Recalled Echo MRI sequence was used to map spatial and temporal changes in the rate of MR signal decay (R2*) in response to L-3,4-dihydroxyphenylalanine (levodopa) in the striatal dopaminergic system of a rhesus monkey unilaterally lesioned with 4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP). R2* decreased significantly in the right (dopamine depleted) putamen and caudate following levodopa. More focal areas of smaller R2* decline were also observed in these structures in the left hemisphere. The observed spatial and temporal patterns of R2* change support the view that the method is monitoring changes in neural activity.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1996

Analysis of noise in phase contrast MR imaging.

In this work we analyze the effects of inherent random noise on the detectability of low-contrast vessel structures that possess slow flow. When flow is encoded in more than one direction, the number of independent noise contributions increases in addition to the scan time. In a fast-flow scenario, only the noise contribution from sampling along the direction of flow is of any significance. At slow flow rates, however, it becomes necessary to account for the noise in each encoded Cartesian direction. The degree to which noise affects low-contrast detectability also depends on the method of phase contrast image processing employed. A theoretical analysis of the statistical properties of signal and noise in processed phase contrast magnitude images is presented and verified from experimental MR image data. Results show a progressively increased bias in the processed phase contrast image magnitude at slow flow rates due to contributions from inherent random noise. The amount of this bias increases with the number of physical directions in which flow is encoded and is larger for complex difference processed images than for phase difference processing. Correspondingly, the output signal-to-noise ratio associated with flow is compromised.

Biometry↗
PubMed1996

Analysis of functional domain organization in DNA topoisomerase II from humans and Saccharomyces cerevisiae.

The functional domain structure of human DNA topoisomerase IIalpha and Saccharomyces cerevisiae DNA topoisomerase II was studied by investigating the abilities of insertion and deletion mutant enzymes to support mitotic growth and catalyze transitions in DNA topology in vitro. Alignment of the human topoisomerase IIalpha and S. cerevisiae topoisomerase II sequences defined 13 conserved regions separated by less conserved or differently spaced sequences. The spatial tolerance of the spacer regions was addressed by insertion of linkers. The importance of the conserved regions was assessed through deletion of individual domains. We found that the exact spacing between most of the conserved domains is noncritical, as insertions in the spacer regions were tolerated with no influence on complementation ability. All conserved domains, however, are essential for sustained mitotic growth of S. cerevisiae and for enzymatic activity in vitro. A series of topoisomerase II carboxy-terminal truncations were investigated with respect to the ability to support viability, cellular localization, and enzymatic properties. The analysis showed that the divergent carboxy-terminal region of human topoisomerase IIalpha is dispensable for catalytic activity but contains elements that specifically locate the protein to the nucleus.

Amino Acid Sequence↗
PubMed1993

Cloning and expression of a novel human profilin variant, profilin II.

We have isolated a 1.7 kbp cDNA encoding a 140 amino acid protein (15.1 kDa, pI 5.91) with a high sequence similarity (62%) to human profilin (profilin I). We have termed this variant profilin II. Northern blot analysis showed that profilin II is highly expressed in brain, skeletal muscle and kidney and less strongly in heart, placenta, lung and liver. In addition, three different transcript lengths were detected. Only one transcript of profilin I was found. The expression level of this was low in brain and skeletal muscle, medium in heart and high in placenta, lung, liver and kidney.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and expression of the transformation sensitive epithelial marker stratifin. A member of a protein family that has been involved in the protein kinase C signalling pathway.

We have identified a family of abundant acidic human keratinocyte proteins with apparent molecular masses ranging between 30,000 and 31,100 (isoelectric focussing sample spot proteins 9109 (epithelial marker stratifin), 9124, 9125, 9126 and 9231 in the master two-dimensional gel database of human keratinocyte proteins) that share peptide sequences with each other, with protein 14-3-3 and with the kinase C inhibitory protein. Immunofluorescence staining of keratinocytes showed that two of these proteins (IEF SSPs 9124 and 9126) localize to the Golgi apparatus, while stratifin is distributed diffusely in the cytoplasm. Significant levels of stratifin, and in smaller amount the sample spot proteins 9124, 9125 and 9126, were detected in the medium of cultured human keratinocytes suggesting that they are partially secreted by these cells. Two-dimensional gel analysis of proteins from cultured human cells and fetal tissues showed that polypeptides comigrating with proteins 9124, 9125 and 9126 are ubiquitous and highly expressed in the brain. Stratifin, however, was present only in cultured epithelial cells and was most abundant in fetal and adult human tissues enriched in stratified squamous keratinising epithelium. We have cloned and sequenced cDNAs coding for members of this family. The complete identity of the sequenced peptides from stratifin with the amino acid sequence translated from the stratifin cDNA clone indicated that this cDNA codes for stratifin. The identity of clones 1054, HS1 and AS1 is less clear as, with few exceptions, none of the individual peptide sequences fits the predicted protein sequences. The polypeptides synthesized by clones 1054 and HS1 in the vaccinia expression system, on the other hand, comigrate with proteins 9126 and 9124, suggesting cell-type-specific expression of members of the protein family. Database searches indicated that clone HS1 corresponds to a human T-cell cDNA 14-3-3 clone, while the high level of similarity of clones 1054 and AS1 with the 14-3-3 beta and eta sequences respectively, suggested that they code for the human equivalent of the two bovine proteins. Microsequence data indicated that IEF SSP 9124 corresponds to the human homolog of bovine 14-3-3 gamma.

14-3-3 Proteins↗
PubMed1993

Eukaryotic topoisomerase I-mediated cleavage requires bipartite DNA interaction. Cleavage of DNA substrates containing strand interruptions implicates a role for topoisomerase I in illegitimate recombination.

Topoisomerase I-mediated cleavage has previously been demonstrated to require interaction of the enzyme with a DNA duplex region encompassing the cleavage site (Svejstrup, J. Q., Christiansen, K., Andersen, A. H., Lund, R., and Westergaard, O (1990) J. Biol. Chem. 265, 12529-12535). The required region, designated region A, includes positions -5 through -1 on the noncleaved strand and positions -7 through +2 on the scissile strand, relative to the cleavage site. Utilizing defined DNA substrates in topoisomerase I cleavage assays we show that efficient cleavage within region A requires additional interaction of the enzyme with duplex DNA on the side holding the 5'-OH end generated by cleavage. By analyzing the interaction of topoisomerase I with DNA substrates varying by single nucleotides on either strand outside region A, an additional duplex region, designated region B, was delimited to positions 6-11. The ability of topoisomerase I to interact separately with regions A and B was assayed on sets of DNA substrates containing a nested series of single-stranded branch sites. The obtained results demonstrate that the normal reversible cleavage/religation equilibrium established by topoisomerase I on continuous duplex DNA is replaced by irreversible cleavage on DNA substrates containing branch sites between the cleavage site and region B as these DNA substrates allow cleavage but prevent religation due to release of the incised strands. The intramolecular bipartite interaction mode of topoisomerase I during the cleavage reaction is thus indicated by both the absence of enzyme-mediated duplex stabilization and the wide tolerance for protruding strands between the cleavage site and region B. Since the irreversibly cleaved topoisomerase I-DNA complexes are kinetically competent to ligate added DNA fragments carrying free 5'-OH ends, the results suggest a role of topoisomerase I in illegitimate recombination.

Base Sequence↗
PubMed1993

The sequence of 28S ribosomal RNA varies within and between human cell lines.

The primary structure of 28S ribosomal RNA constitutes a conserved core which is similar among most 23S-like rRNAs and expansion segments which occur at specific positions in the sequence. The expansion segments account for most of the size difference between prokaryotic (archaeal and eubacterial) and eukaryotic rRNAs and they exhibit a sequence variation which is unique among rRNAs. We have investigated the sequence variation of one of the expansion segments, V8, by sequencing a total of 111 V8 segments from 9 different human cell lines and tissues and have found 35 different variants. The variation occur mainly at two 'hot spots' which are separated by 170 nucleotides in the primary sequence but are neighbours in the secondary structure. The sequence of V8 segments varies both within and between human cell lines and tissues. The implications for the evolution of the eukaryotic 28S rRNA are discussed together with possible functions of the expansion segments. We also present a secondary structure model for the V8 segment based on comparative sequence analysis and chemical and enzymatic foot printing.

Base Sequence↗
PubMed1993

Identification of two human Rho GDP dissociation inhibitor proteins whose overexpression leads to disruption of the actin cytoskeleton.

Proteins (IEF's 1120, 8118, 8120) sharing similarity to the bovine Rho GDP dissociation inhibitor (GDI) have been identified in the human two-dimensional-gel database of keratinocyte proteins. Molecular cloning of the corresponding cDNAs showed that IEF 8118 is the human homolog of bovine GDI while IEF 8120 is a distinct although related protein. All available information indicates the IEF 1120 is a derivative of IEF 8120. The cDNAs coding for IEF's 8118 and 8120 were recombined into vaccinia virus and expressed in differentiated human keratinocytes and their effect on the actin cytoskeleton was assessed by immunofluorescence using TRITC-phalloidin. The results showed that overexpression of both GDI proteins leads to rounding up of the cells and loss of stress fibers and focal contact sites. In addition, the cell to cell adhesion belts gradually disappeared, an effect that was particularly pronounced in infected cells overexpressing IEF 8120. Taken together, the results imply that Rho GDI's play a role in modulating the activity of the Rho proteins as their overexpression mimics phenotypic changes associated with the inactivation of these proteins.

Actins↗
PubMed1992

Mode of action of topoisomerase II-targeting agents at a specific DNA sequence. Uncoupling the DNA binding, cleavage and religation events.

Methods of uncoupling the DNA binding, cleavage and religation reactions of topoisomerase II were employed to investigate the influence of topoisomerase II-directed drugs on the individual steps in the enzyme's catalytic cycle. A special DNA substrate containing a major topoisomerase II interaction site, which can be cleaved by the enzyme in the absence of any concomitant religation, was used to examine the effect of topoisomerase II-directed agents upon the DNA cleavage reaction. The experiment demonstrated that the topoisomerase II targeting agent Ro 15-0216 stimulates the DNA cleavage reaction extensively, whereas the traditional topoisomerase II inhibitor, mAMSA, has only a minor effect on this reaction. Topoisomerase II trapped in the cleavage complexes can religate to the 3' hydroxyl end of another DNA strand. Using this religation assay, it was demonstrated that the major effect of mAMSA is an inhibition of the enzyme's religation reaction, whereas Ro 15-0216 has no effect on this reaction. Recently, considerable attention has been given to drugs preventing topoisomerase II from introducing DNA cleavages. In the present paper the initial non-covalent DNA binding reaction of topoisomerase II was investigated under conditions excluding enzyme-mediated DNA cleavage. This demonstrated that the anthracycline, aclarubicin, prevents topoisomerase II from performing its initial non-covalent DNA binding reaction and thereby abolishes the DNA cleavage reaction of the enzyme. The results presented here demonstrate that profound differences exist in the mode of action of different agents targeting topoisomerase II, and that the enzyme can be affected by such agents at both its DNA binding, cleavage and religation subreactions.

Acetanilides↗
PubMed1992

8-methoxycaffeine inhibition of Drosophila DNA topoisomerase II.

We have investigated the effect of 8-methoxycaffeine on the interaction between Drosophila DNA topoisomerase II and DNA. We have shown that 8-methoxycaffeine affected the enzyme strand-passing activity by inhibiting decatenation of kinetoplast DNA, and that it interfered with the breakage-reunion reaction by stabilizing a cleavable complex. Treatment of the cleavable complex with protein denaturant resulted in DNA breaks. High resolution mapping of the cleavage sites in the central spacer region of Tetrahymena rDNA revealed that, contrary to what was observed with clinically important DNA topoisomerase II inhibitors, 8-methoxycaffeine did not modify the cleavage pattern observed without the drug.

Animals↗
PubMed1991

New technique for uncoupling the cleavage and religation reactions of eukaryotic topoisomerase I. The mode of action of camptothecin at a specific recognition site.

A new technique for uncoupling the cleavage and religation half-reactions of topoisomerase I at a specific site has been developed. The technique takes advantage of a suicidal DNA substrate to attain enzyme-mediated cleavage without concomitant religation. Efficient religation can be achieved, subsequently, by addition of an oligonucleotide capable of hybridising to the non-cleaved strand of the suicide DNA substrate. The technique was used to study the effect of different compounds on the half-reactions of topoisomerase I. It was shown that topoisomerase I-mediated cleavage was inhibited by NaCl concentrations higher than 200 mM, while the religation reaction seemed unaffected by concentrations as high as 3 M-NaCl. The divalent cations Mg2+, Ca2+ and Mn2+ were found to enhance the cleavage but not the religation reaction of topoisomerase I, whereas Cu2+ and Zn2+ inhibited both reactions. Furthermore, the effect of the anti-neoplastic agent, camptothecin, on the half-reactions of topoisomerase I was investigated. It was found that the drug did not affect the cleavage reaction of topoisomerase I at the studied site, while the religation reaction of the enzyme was inhibited. Camptothecin was found to stabilise the enzyme-DNA cleavage complex even when the drug was added after complex formation.

Antineoplastic Agents↗
PubMed1991

Drug Stimulated DNA Cleavage Mediated by Cauliflower Topoisomerase II.

We have investigated cauliflower (Brassica oleracea) topoisomerase II with respect to its interaction with DNA and demonstrate that the enzyme shares the characteristics of topoisomerase II purified from a variety of phylogenetically remote organisms. In the presence of the 2-nitroimidazole Ro 15-0216, cauliflower topoisomerase II-mediated DNA cleavage is extensively stimulated (approximately 20-fold) only at a site recognized as a major cleavage site for the enzyme in the absence of drug. The conservation of the enzyme's DNA specificity in the presence of Ro 15-0216 is in contrast to the effect exerted by traditional topoisomerase II inhibitors, which cause enzyme-mediated cleavage to take place at a multiple number of DNA sites. Ro 15-0216 may therefore prove useful as a tool in the elucidation of the enzyme's DNA interaction sites and its involvement in nucleic acid metabolism in plant cells.

Journal Article↗