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L M Fisher

Publications and source records attributed to L M Fisher.

At least 73 records · Page 4Linked to original sources
PubMed1990

Isolation and characterization of a human cDNA clone encoding a novel DNA topoisomerase II homologue from HeLa cells.

We have isolated and sequenced 3 human DNA topoisomerase II (topo II) partial cDNA clones from a HeLa carcinoma cell cDNA library. Two clones were identical to an internal fragment of HeLa topo II cDNA. The third clone, CAA5, had a different and novel sequence which shared significant nucleotide (62%) and predicted peptide (70%) homologies with a region of the HeLa topo II cDNA. Our results suggest that HeLa cells express at least two homologous forms of DNA topoisomerase II. The new HeLa topo II homologue is discussed in relation to topo II isoenzymes recently described in a Burkitt lymphoma and other cell lines.

Amino Acid Sequence↗
PubMed1990

DNA binding and intercalation by novel porphyrins: role of charge and substituents probed by DNase I footprinting and topoisomerase I unwinding.

Porphyrins carrying four charged sidechains, e.g., meso-tetrakis[4-N-methylpyridiniumyl]- and meso-tetrakis[4-N-(2-hydroxyethyl)pyridiniumyl]-porphyrin, bound and intercalated similarly into DNA as measured by helix stabilization and DNA unwinding studies in the presence of DNA topoisomerase I. Despite their different bulky sidechains, these complexes gave essentially identical DNase I footprinting patterns. In contrast, tetrasubstituted porphyrins carrying three phenyl rings and a single positively charged pyridiniumyl sidechain did not intercalate and exhibited little affinity for DNA. Thus, the presence of charged sidechains on the porphyrin rather than their identity appears to be critical for efficient DNA intercalation. The results are discussed in regard to current models for the porphyrin-DNA intercalation complex.

Base Sequence↗
PubMed1990

Characteristics of telephone applicants to a residential rehabilitation program for homeless veterans.

This study gathered descriptive data on 163 individuals who applied by telephone to a residential rehabilitation program for homeless veterans and compared these data with general veteran and homeless populations. Ss were a young (M = 40.82 years) and educated (M = 13.34 years of schooling) subgroup of homeless men (98.16%) with histories of relatively high, stable functioning (e.g., previous successful employment) and high rates of medical (47.47%), substance abuse (67.1%), psychiatric (41.93%; primarily nonpsychotic), and legal (71.15%) problems. These characteristics appear to be different from those of other subgroups of homeless (e.g., homeless chronically mentally ill, skid-row alcoholics), and they provide a basis for beginning to develop distinct remedial strategies that are specific to this subpopulation. The advantages of studying subgroups of homeless and the utility of the telephone interview data collection methodology are discussed.

Adult↗
PubMed1990

Stroop Color-Word Test performance in patients with Alzheimer's disease.

Patients with Alzheimer's disease (AD) (n = 36) and normal older adults (n = 36) were individually administered the Stroop Color-Word Test. Eight of 36 (22%) AD patients exhibited confusion between the colors blue and green, while no control subject had difficulty distinguishing among the colors. In a second experiment, a subset of the original sample (15 AD patients and 8 control subjects) was retested using the Stroop. Only 2 AD patients showed color confusion on both test occasions, while 7 AD patients exhibited color confusion on one occasion. No control subject exhibited confusion between colors the second time. These results indicate that color confusion in AD patients is inconsistent. Due to the high incidence of color confusion in AD patients, the Stroop should be used with caution in patient populations.

Aged↗
PubMed1990

DNA gyrase gyrA mutations in ciprofloxacin-resistant strains of Staphylococcus aureus: close similarity with quinolone resistance mutations in Escherichia coli.

The gyrA genes isolated from three ciprofloxacin-resistant clinical isolates of Staphylococcus aureus carried codon 84 (serine----leucine) and/or codon 85 (serine----proline) mutations that were absent in pretreatment susceptible strains. These substitutions occur in a region of the gyrase A protein wherein directly analogous mutations of serine 83----leucine and alanine 84----proline in Escherichia coli confer quinolone resistance. Thus, DNA gyrase A subunit mutations are implicated in resistance to ciprofloxacin in S. aureus.

Amino Acid Sequence↗
PubMed1990

DNA cloning and organization of the Staphylococcus aureus gyrA and gyrB genes: close homology among gyrase proteins and implications for 4-quinolone action and resistance.

Staphylococcus aureus gyrA and gyrB genes, which encode the DNA gyrase A and B proteins, have been isolated and found to map contiguously. DNA sequence analysis revealed close homology between the S. aureus gyrase subunits and their counterparts in Bacillus subtilis and Escherichia coli, including several conserved amino acid residues whose substitution in E. coli confers resistance to 4-quinolones. These results are discussed in regard to quinolone resistance mechanisms in S. aureus.

Amino Acid Sequence↗
PubMed1990

Development and properties of an etoposide-resistant human leukaemic CCRF-CEM cell line.

Etoposide (VP-16) and several other unrelated anti-tumour agents appear to act by inhibiting the enzyme DNA topoisomerase II. We report here the development and characterization of an etoposide-resistant human leukaemic CCRF-CEM cell line, CEM/VP-1. The cell line was 15-fold more resistant to etoposide than the parental CEM cells and exhibited cross-resistance to other topoisomerase II inhibitors including teniposide, m-AMSA, and doxorubicin. CEM/VP-1 cells exhibited only a low level cross-resistance to the Vinca alkaloids, vinblastine and vincristine, known inhibitors of mitotic spindle formation. As a first step in defining the mechanism of resistance to etoposide, we compared the levels of topoisomerase II activity and its drug sensitivity in nuclear extracts from the resistant and sensitive CEM cells. As determined by a kinetoplast DNA decatenation assay, the level of DNA topoisomerase II activity in CEM/VP-1 nuclear extracts was approximately 2-fold lower than that in CEM cells, and the activity appeared to be resistant to inhibition by etoposide. Furthermore, the DNA topoisomerase II activity in CEM/VP-1 nuclear extracts did not promote the etoposide-dependent cleavage of pBR322 DNA observed with extract from sensitive cells. These results suggest that etoposide resistance in the CEM/VP-1 cell line may at least in part be due to an altered topoisomerase II, or associated factor, resulting in a reduced ability to induce DNA cleavage in the presence of drug.

Cell Division↗
PubMed1990

DNA topoisomerases: enzymes that change the shape of DNA.

The topological state of DNA plays a crucial role in many biological processes including DNA replication and gene expression. DNA topoisomerases are enzymes, present in all cells, that modulate and regulate the topology of DNA, e.g. by introducing or removing DNA supercoils. New insight into the reaction mechanisms and cellular functions of these biologically essential proteins has come from novel enzymatic and genetic approaches. Medical interest in topoisomerases has developed from the discovery that they are the intracellular targets for drugs used in the treatment of infectious diseases and cancer. This review describes mechanistic, biological and medical advances in our understanding of these intriguing proteins.

DNA↗
PubMed1989

Ciprofloxacin and the fluoroquinolones. New concepts on the mechanism of action and resistance.

Ciprofloxacin, a new fluoroquinolone, is a potent, broad-spectrum antibacterial agent. It rapidly blocks bacterial deoxyribonucleic acid (DNA) replication by inhibiting DNA gyrase, an essential prokaryotic enzyme that catalyzes chromosomal DNA supercoiling. Molecular genetic approaches have been used to study the interaction of 4-quinolones with DNA gyrase from quinolone-sensitive strains and from uropathogenic quinolone-resistant clinical isolates of Escherichia coli. An important mutational locus in the gyrase A gene that confers resistance to ciprofloxacin and other quinolones has been identified, and a new, rapid method to examine clinical isolates for the presence of mutations at this position has been devised. A quinolone resistant gyrA gene has been previously cloned and sequenced from an E. coli clinical isolate. Genetic analysis indicated that resistance resulted from a Ser-83----Trp change in the 875 residue gyrase A protein: two other changes observed in the protein, Asp-678----Glu and Ala-828----Ser, were neutral. GyrA genes carrying these mutations have now been expressed, corresponding mutant gyrase A proteins purified, and their quinolone resistance properties tested by complementing with gyrase B protein and studying the resulting gyrase activity in an adenosine triphosphate-dependent DNA supercoiling assay. The in vitro DNA supercoiling activity of the A (Ser-83----Trp) mutant subunit complemented with wild-type gyrase B subunit was highly resistant to ciprofloxacin and other 4-quinolones. In contrast, A subunit carrying codon 678 and 828 changes reconstituted a quinolone-sensitive gyrase activity. Thus, quinolone-resistant gyrase A proteins may be readily obtained for study by using high-copy gyrA plasmids. In addition, other quinolone-resistant strains of E. coli have been examined for the presence of mutations at gyrase A codons 82 and 83 using a new analytical method based on a restriction fragment length polymorphism (RFLP). This analysis revealed that seven of eight resistant clinical isolates of E. coli examined carried gyrA mutations at codon 82 or 83, whereas five sensitive strains appeared to possess wild-type sequence. Thus, mutations at codon 83 (and possibly 82) in the gyrA gene frequently confer resistance to 4-quinolones, including ciprofloxacin. The RFLP method described should prove useful in examining strains for such mutations. These results are discussed with regard to the mode of interaction of the 4-quinolones with gyrase.

Anti-Infective Agents↗
PubMed1989

Regulation of DNA supercoiling in Escherichia coli: genetic basis of a compensatory mutation in DNA gyrase.

Bacterial DNA supercoiling is controlled by balancing the supercoiling activity of DNA gyrase and the relaxing activity of DNA topoisomerase I. We have characterized the gyrB gene from a top A deletion mutant of Escherichia coli (DM800) that has a compensatory mutation in gyrB, lowering the activity of gyrase 10-fold, and thereby redressing the intracellular level of supercoiling. The mutant gene differs from the wild type in carrying three rather than two direct tandem repeats of a 6 bp sequence encoding Ala-Arg. We suggest this novel mutation affects domain spacing and was generated by an unequal crossing over event, possibly involving gyrase.

Base Sequence↗
PubMed1989

Cloning and characterization of a DNA gyrase A gene from Escherichia coli that confers clinical resistance to 4-quinolones.

Nalidixic acid, enoxacin, and other antibacterial 4-quinolones inhibit DNA gyrase activity by interrupting DNA breakage and reunion by A subunits of the A2B2 gyrase complex. Despite their clinical importance, the mode of quinolone action and mechanisms of resistance are poorly understood at the molecular level. Using a DNA fragment enrichment procedure, we isolated the gyrA gene from a uropathogenic Escherichia coli strain that encodes a gyrase A protein cross-resistant to a variety of quinolones. When complemented with gyrase B subunit, the purified A protein reconstituted DNA supercoiling activity approximately 100-fold more resistant to inhibition by enoxacin than the susceptible enzyme and failed to mediate quinolone-dependent DNA cleavage. Nucleotide sequence analysis revealed that the gene differed at 58 nucleotide positions compared with the K-12 gyrA sequence. The 875-amino-acid residue-resistant gyrase A protein differed at three positions from its wild-type E. coli K-12 counterpart: tryptophan, glutamate, and serine replaced serine, aspartate, and alanine residues at positions 83, 678, and 828, respectively. By genetic analysis of chimeric gyrA genes in a gyrA(Ts) background, we showed that the Ser-83----Trp mutation in the gyrase A protein was solely responsible for high-level bacterial resistance to nalidixic acid and fluoroquinolones.

4-Quinolones↗
PubMed1986

Energetics of proline racemase: racemization of unlabeled proline in the unsaturated, saturated, and oversaturated regimes.

The interconversion of L- and D-proline catalyzed by proline racemase has been studied. The entire time course of the approach to equilibrium has been followed. After a short time the product concentration is significant, and the reaction runs under reversible conditions. As the total substrate concentration is increased, the system moves from the unsaturated regime into the saturated regime. At very high substrate levels under the reversible conditions used, the rate constant for substrate racemization falls, as the system moves into the "oversaturated" regime. Here, the net rate of the enzyme-catalyzed reaction is limited by the rate of return of the free enzyme from the form that liberates product back to the form that binds substrate. The results are analyzed in terms of the simple mechanism (table; see text) and illustrate the additional information that is available from reactions studied under reversible conditions. In the unsaturated region the value of the second-order rate constant kU (equivalent to kcat/Km) is 9 X 10(5) M-1 s-1 in each direction. In the saturated region, kcat = kcat = 2600 s-1 and Km = 2.9 mM. In the oversaturated region, the rate constant kO is 81 M s-1. The substrate concentration at which unsaturated and saturated terms contribute equally is 2.9 mM, and the substrate concentration at which saturated and oversaturated terms contribute equally is 125 mM.

Amino Acid Isomerases↗
PubMed1986

Energetics of proline racemase: tracer perturbation experiments using [14C]proline that measure the interconversion rate of the two forms of free enzyme.

Proline racemase exists in two states, one of which binds and isomerizes L-proline and the other of which binds and isomerizes D-proline. In the enzyme-catalyzed racemization of proline at high substrate concentrations, the interconversion of the two forms of the free enzyme becomes rate limiting. The tracer perturbation method of Britton (1966, 1973) vividly demonstrates the kinetic importance of this enzyme interconversion under oversaturating conditions and allows an estimate of the rate constant for this reaction of 10(5) s-1. It is further shown that the enzyme is bound state saturated and the peak-switch concentration, Cp, is 125 mM. At substrate concentrations higher than 125 mM the enzyme becomes oversaturated, and the reaction rate is limited by the transition state for the interconversion of two forms of the free enzyme. It seems likely that the two free enzyme forms differ only in the protonation states of the acidic and basic groups at the active site.

Amino Acid Isomerases↗
PubMed1986

Energetics of proline racemase: transition-state fractionation factors for the two protons involved in the catalytic steps.

The isotope effects for the interconversion of L-proline and D-proline, catalyzed by proline racemase, have been determined in the saturated region with both [2-2H]proline and [2-3H]proline. The deuterium fractionation factors for each of the protons in flight have been obtained from two kinds of experiment: by measuring the rate of racemization of one [2-2H]proline enantiomer as it racemizes into an equilibrated pool of unlabeled proline and by measuring the deuterium content of a proline sample at the optical rotation maximum that occurs when an equimolar mixture of one deuterium-labeled enantiomer and the other unlabeled enantiomer runs to equilibrium. The tritium fractionation factors for each of the protons in flight have been determined from measurements of the rate of loss of tritium to the solvent as one [2-3H]proline enantiomer runs to equilibrium. Good agreement is found among the fractionation factors determined by each method. The deuterium fractionation factors for the two protons are not identical: that for the proton derived from L-proline is 0.375 and that for the proton derived from D-proline is 0.44. This difference has been confirmed by a double-competition experiment in which the optical rotation of a mixture of DL-[2-2H]proline and unlabeled DL-proline is followed with time. The rotation (initially zero) passes through a maximum, from which the ratio of the two fractionation factors (0.86) is obtained. These data, coupled with the equilibrium fractionation factor for the 2-position of proline (which has been determined to be 1.17), provide the transition-state factors for each of the in-flight protons, and delineate the nature of the transition state(s) for the enzyme-catalyzed racemization.

Amino Acid Isomerases↗
PubMed1986

Energetics of proline racemase: rates, fractionation factors, and buffer catalysis in the oversaturated region. Nature of the interconversion of the two forms of free enzyme.

To probe the nature of the interconversion of the two unliganded forms of proline racemase, a number of experiments have been performed under oversaturating conditions where the rate of the enzymic reaction is mainly limited by the rate of this interconversion. Competitive deuterium washout experiments, where an equimolar mixture of D- and L-proline (in which some or all of one enantiomer is specifically deuterated at the 2-position) is allowed to reach chemical and isotopic equilibrium mediated by the enzyme, have been followed in four ways. The size and the rate of achievement of the maximum perturbation in the optical rotation have been measured, the deuterium content of the substrate at this maximum has been determined, and the final approach to equilibrium after the perturbation maximum has been followed. Further, the enzyme-catalyzed rate of tritium loss from [2-3H]proline has been established. Finally, it has been shown that the enzyme interconversion reaction is catalyzed by several buffers (such as ammonium, hydrazinium, and hydrogen sulfide). These data are discussed in terms of Marcus' theory, which allows a rather detailed picture of the mechanism of free enzyme interconversion to be drawn. This process nicely parallels the mechanism of the enzyme-catalyzed interconversion of the proline enantiomers, and it is evident that substrate racemization (with the concomitant switch of the enzyme-bound protons) is mirrored by the water-mediated switch of the enzyme-bound protons that effects the interconversion of the free enzyme forms. The results favor a stepwise reaction for the interconversion of the free enzyme forms in which a proton is abstracted from a bound water molecule to give a reaction intermediate having a hydroxide ion bound to the diprotonated form of the enzyme.

Amino Acid Isomerases↗
PubMed1986

DNA gyrase complex with DNA: determinants for site-specific DNA breakage.

DNA gyrase catalyses DNA supercoiling by making a transient double-stranded DNA break within its 120-150 bp binding site on DNA. Addition of the inhibitor oxolinic acid to the reaction followed by detergent traps a covalent enzyme-DNA intermediate inducing sequence-specific DNA cleavage and revealing potential sites of gyrase action on DNA. We have used site-directed mutagenesis to examine the interaction of Escherichia coli gyrase with its major cleavage site in plasmid pBR322. Point mutations have been identified within a short region encompassing the site of DNA scission that reduce or abolish gyrase cleavage in vitro. Mapping of gyrase cleavage sites in vivo reveals that the pBR322 site has the same structure as seen in vitro and is similarly sensitive to specific point changes. The mutagenesis results demonstrate conclusively that a major determinant for gyrase cleavage resides at the break site itself and agree broadly with consensus sequence studies. The gyrase cleavage sequence alone is not a good substrate, however, and requires one or other arm of flanking DNA for efficient DNA breakage. These results are discussed in relation to the mechanism and structure of the gyrase complex.

Base Sequence↗