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Biomedical subjects

L M Fisher

Publications and source records attributed to L M Fisher.

At least 55 records · Page 3Linked to original sources
PubMed1995

Expression in Escherichia coli and purification of human eosinophil-derived neurotoxin with ribonuclease activity.

Eosinophil-derived neurotoxin (EDN) is a ribonuclease with neurotoxic and helminthotoxic properties. It is present in the crystalloid granules of human eosinophils. We report the expression and characterization of a functionally active recombinant human EDN using the pMAL-cRI expression system. A cDNA for mature EDN was obtained by PCR and inserted in pMAL-cRI downstream of the malE gene encoding maltose binding protein. Induction of the ptac promoter of the plasmid in Escherichia coli strain BL21(DE3) resulted in high level expression of soluble MAL-EDN fusion protein. Cleavage of affinity purified fusion protein with Factor Xa protease released recombinant EDN which comigrated with native EDN on SDS-polyacrylamide gels and cross-reacted with a polyclonal anti-EDN antiserum on Western blots. IN contrast to previous attempts at EDN expression, denatured and refolded EDN had ribonuclease activity and was prepared in microgram amounts. The availability of recombinant human EDN should facilitate studies of its structure and biological functions.

ATP-Binding Cassette Transporters↗
PubMed1995

Aging and speed of behavior: possible consequences for psychological functioning.

Over 100 years of observations have established that slowness of behavior is a characteristic of becoming old, although it is now recognized that health, use of medications, and physical activity may modify the extent of the slowing. Early research indicated that there is a limited contribution to slowing by peripheral sensory-motor factors. Substantial evidence has pointed to the central nervous system as the locus of the slowing. Recent investigators have expressed divided opinions about whether there is a pervasive general slowing of behavior by the central nervous system or whether there are specific localized mechanisms. This is not unlike early disputed views of the brain as having localized or global behavioral functions: Both principles appear to be simultaneously true. Sufficient research has been conducted to indicate that there are specific factors as well as a general process associated with the slowing of behavior with advancing age. Whether such slowing is a primary or secondary cause of age differences in cognitive processes is a significant scientific issue. A marked broadening of research on aging has been accompanied by an interest in identifying both the neurophysiological correlates of slowing as well as its role in specific cognitive processes. Yet another aspect of the changing research picture is the trend to move beyond the mere use of chronological age as the sole basis for comparing performance differences. Measurement of more independent variables is suggested as part of clusters or causal complexes that will indicate sources of the changes in speed and other aspects of behavior. These causal complexes include biological indicators such as disease, physiological capacity for work, and length of life, as well as causal complexes of social factors involving such variables as education, occupation, and ethnicity. There has been considerable discussion of markers of aging. In this approach, factors found to be closely associated with advancing age are used as measures of the effectiveness of attempts to modify the course of aging, e.g. by diet, exercise, new learning, and drugs. Along with other biomarkers of aging, speed of behavior may prove to be a criterion for assessing the impact of interventions on the rate and processes of aging. As a marker of aging, speed needs further exploration that will compare the slowness observed in different subgroups of adults with a wide range of outcomes in their productivity, capacity for adaptation to life's demands, and health. The present status of information about slowness of behavior with advancing age indicates that it is one of the most reliable features of human life.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1993

Use of yeast in the study of anticancer drugs targeting DNA topoisomerases: expression of a functional recombinant human DNA topoisomerase II alpha in yeast.

A plasmid was constructed for the expression of human DNA topoisomerase II alpha in yeast from a galactose-inducible promoter of the yeast GAL1 gene. Expression of a recombinant human enzyme, in which the first 28 of the 1531 codons of human DNA topoisomerase II alpha were replaced by the first five codons of yeast DNA topoisomerase II, was shown to rescue the lethal phenotype of thermal sensitive yeast DNA topoisomerase II mutants at 35 degrees C. Purification of the human enzyme overexpressed in yeast yielded a single polypeptide with an apparent mass of 170 kDa, and the properties of the purified recombinant enzyme were found to be the same as those reported for human DNA topoisomerase II alpha purified from HeLa cells. Studies with the anticancer drug amsacrine indicated that the human enzyme, either inside yeast cells or in its purified form, is a target of the drug; inhibition of the purified enzyme by teniposide (VM-26) and merbarone was also demonstrated. These studies demonstrate that yeast strains expressing human DNA topoisomerase II alpha provide a convenient system for studying drugs targeting the enzyme; unlike mammalian systems, potential complications due to the presence of human DNA topoisomerase II beta can be eliminated in this system. Overexpression of human DNA topoisomerase II alpha in yeast also provides a convenient source of the enzyme for in vitro studies.

Antineoplastic Agents↗
PubMed1993

Novel HeLa topoisomerase II is the II beta isoform: complete coding sequence and homology with other type II topoisomerases.

DNA topoisomerase (topo) II mediates DNA strand passage in an ATP-dependent reaction. Human cell lines express at least two genetically distinct forms of the enzyme, topo II alpha (p170) and II beta (p180). Previously, we isolated a novel HeLa cDNA clone (CAA5) that partially encodes a protein homologous to topo II alpha (Austin, C.A. and Fisher, L.M. (1990) FEBS Lett. 266, 115-117). In this paper we show that CAA5 encodes a C-terminal segment of human topo II beta. We report here for the first time cDNA clones spanning the entire coding sequence. Overlapping clones specifying the 3' end of the cDNA have been isolated, mapped and sequenced. The missing 5' coding sequence was obtained by an inverse PCR protocol and from a specifically primed cDNA library. Human topo II beta is a 1621 amino acid protein which is closely homologous to topo II alpha in the N-terminal three quarters of its sequence. In contrast, the C-terminal segments of the alpha and beta sequences show considerable divergence suggesting these regions may mediate different cellular functions of the two isoforms. Southern blot analysis of yeast and Drosophila DNA using human alpha and beta specific probes detected a single topo II homologue in these lower eukaryotes. Comparison of the protein sequence for human topo II beta with other type II topoisomerases revealed several conserved motifs and has allowed identification of the likely ATPase- and DNA breakage-reunion domains.

Base Sequence↗
PubMed1993

Novel selection and genetic characterisation of an etoposide-resistant human leukaemic CCRF-CEM cell line.

We have studied the genetic alterations acquired during selection of a cloned human leukaemic cell line (CEM/VP-1) that is 15-fold more resistant to the anticancer topoisomerase II-inhibitor etoposide than parental CCRF-CEM cells. CEM/VP-1 cells exhibit an 'atypical MDR' phenotype: cross resistance to other topo II inhibitors (but not Vinca alkaloids) and expression of a drug-resistant topo II activity. Cytogenetic and molecular studies revealed that the cell line carried multiple genetic changes affecting TOP2 genes encoding both topo II alpha and beta isoforms. CEM/VP-1 was diploid, 47,XX,+20, and appears to have been preferentially selected from a 1% diploid subpopulation present in the tetraploid parental cells. The same chromosomal abnormalities were present in resistant and sensitive cells except for an acquired 3p- change most likely deleting one TOP2 beta allele. PCR/DNA sequence analysis and allele-specific hybridisation showed that one of two TOP2 alpha alleles expressed in CEM/VP-1 cells had acquired a Lys-797-->Asn codon change. This mutation lies close to the catalytic Tyr-804 residue of the protein and may interfere with drug-induced trapping of the cleavable complex. Alternatively, it could exert a loss of function phenotype. CEM/VP-1 cells did not exhibit codon 449 or 486 TOP2 alpha mutations in the ATP binding domain reported in two other resistant cell lines. Diploid selection and multiple changes observed in CEM/VP-1 cells appear to be consequences of the recessive phenotype of at-MDR. These results may be useful in approaching the mechanisms of clinical resistance.

Base Sequence↗
PubMed1993

Item and relational processing in young and older adults.

The item and relational information framework of memory provided a methodology for an analytical approach to age differences in encoding and retrieval processes. Subjects were given related or unrelated words to sort or rate for pleasantness. Young adults performed better than older adults in free recall, but older adults were able to use both item and relational information as well as young adults. In a second experiment, subjects also sorted or rated a list composed of different numbers of category items. The pattern of free recall and cued recall scores indicated that item and relational information affected the memory performance of both age groups in a similar fashion. By examining memory in terms of item and relational information, it appears that older adults do not have difficulty combining two types of target information during encoding and that the information assists in recall of target items.

Adolescent↗
PubMed1992

Escherichia coli DNA gyrase: genetic analysis of gyrA and gyrB mutations responsible for thermosensitive enzyme activity.

Escherichia coli gyrA43 and gyrB203 alleles conferring temperature-sensitive (ts) growth encoded Gly751-->Asp and Pro171-->Ser substitutions in the DNA gyrase A and B subunits, respectively. A plasmid-borne gyrA43 allele was genetically dominant over a chromosomal quinolone-resistant gyrA gene at 30 degrees C but not at 42 degrees C. These results and others confirm the ts phenotype of the mutation, the first to be identified in the C-terminal DNA binding/complex stabilizing domain of gyrase A protein. By contrast, the Pro171-->Ser mutation is located near the ATP-binding site of gyrase B protein and could interfere with energy coupling during DNA supercoiling. These data are discussed in regard to recently described gyrA(ts) mutations that affect the control of chromosome segregation.

Alleles↗
PubMed1992

DNA supercoiling and relaxation by ATP-dependent DNA topoisomerases.

Bacterial DNA gyrase and the eukaryotic type II DNA topoisomerases are ATPases that catalyse the introduction or removal of DNA supercoils and the formation and resolution of DNA knots and catenanes. Gyrase is unique in using ATP to drive the energetically unfavourable negative supercoiling of DNA, an example of mechanochemical coupling: in contrast, eukaryotic topoisomerase II relaxes DNA in an ATP-requiring reaction. In each case, the enzyme-DNA complex acts as a 'gate' mediating the passage of a DNA segment through a transient enzyme-bridged double-strand DNA break. We are using a variety of genetic and enzymic approaches to probe the nature of these complexes and their mechanism of action. Recent studies will be described focusing on the role of DNA wrapping on the A2B2 gyrase complex, subunit activities uncovered by using ATP analogues and the coumarin and quinolone inhibitors, and the identification and functions of discrete subunit domains. Homology between gyrase subunits and the A2 homodimer of eukaryotic topo II suggests functional conservation between these proteins. The role of ATP hydrolysis by these topoisomerases will be discussed in regard to other energy coupling systems.

Adenosine Triphosphate↗
PubMed1992

Site-specific DNA cleavage by mammalian DNA topoisomerase II induced by novel flavone and catechin derivatives.

Four naturally occurring flavones (baicalein, quercetin, quercetagetin and myricetin) and two novel catechins [(-)-epicatechin gallate and (-)-epigallocatechin gallate, from the tea plant Camellia sinensis], which are known inhibitors of reverse transcriptase, were shown to induce mammalian topoisomerase II-dependent DNA-cleavage in vitro. The flavones differed from the catechins in causing unwinding of duplex DNA, but both classes of compound induced enzymic DNA breakage at the same sites on DNA. Moreover, the cleavage specificity was the same as that for the known intercalator 4'-(acridin-9-ylamino)methanesulphon-m-anisidide, suggesting that these agents trap the same cleavable complex. Analysis of some 30 flavonoid compounds allowed elucidation of the structure-function relationships for topoisomerase II-mediated DNA cleavage. For flavonoid inhibitors an unsaturated double bond between positions 2 and 3 of the pyrone ring and hydroxy groups at the 5, 7, 3' and 4' positions favoured efficient cleavage. Hydroxy substitutions could be tolerated at the 3, 6 and 5' positions. Indeed, the absence of substituents at the 3', 4' and 5' positions could be compensated by a hydroxy group at position 6 (baicalein). Similar requirements have been reported for flavonoid inhibitors of protein kinase C that act competitively with ATP, suggesting interaction with a conserved protein feature. Formation of the cleavable complex is a cytotoxic lesion that may contribute to the growth-inhibitory properties of flavones observed for three human tumour cell lines. These results are discussed in regard to the selectivity of antiviral agents.

Animals↗
PubMed1992

An Escherichia coli DNA topoisomerase I mutant has a compensatory mutation that alters two residues between functional domains of the DNA gyrase A protein.

Nucleotide sequence analysis revealed that the compensatory gyrA mutation in Escherichia coli DM750 affects DNA supercoiling by interchanging the identities of Ala-569 and Thr-586 in the DNA gyrase A subunit. These residues flank Arg-571, a site for trypsin cleavage that splits gyrase A protein between DNA breakage-reunion and DNA-binding domains. The putative interdomain locations of the DM750 mutation and that of E. coli DM800 (in gyrase B protein) suggests that these compensatory mutations may reduce DNA supercoiling activity by altering allosteric interactions in the gyrase complex.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequence of the Staphylococcus aureus gyrB-gyrA locus encoding the DNA gyrase A and B proteins.

We have determined the nucleotide sequence of a 5.3-kb segment of the Staphylococcus aureus chromosome that includes the gyrA and gyrB genes coding for both subunits of DNA gyrase, the enzyme that catalyzes ATP-dependent DNA supercoiling. The gene order at this locus, dnaA-dnaN-recF-gyrB-gyrA, is similar to that found in the Bacillus subtilis replication origin region. S. aureus recF, gyrB, and gyrA genes are closely spaced, occupy the same reading frame, and may be coordinately expressed. The S. aureus gyrB and gyrA genes encode 640- and 889-residue proteins, respectively, that share strong homology with other bacterial gyrase subunits, notably those from B. subtilis. These results are discussed in regard to the mechanism of DNA gyrase and its role as a target for the 4-quinolones and other antistaphylococcal agents.

Amino Acid Sequence↗
PubMed1991

gyrA mutations in ciprofloxacin-resistant, methicillin-resistant Staphylococcus aureus from Indiana, Minnesota, and Tennessee.

Mutational changes occurring at amino acid codons 84 and 85 located in the gyrA gene of methicillin-resistant Staphylococcus aureus (MRSA) were studied using radiolabeled oligonucleotide probes to examine the incidence of these ciprofloxacin resistance determinants in 30 MRSA isolates from Indiana, Minnesota, and Tennessee. Four separate oligonucleotide probes, one each corresponding to the wild-type sequence, a mutation at codon 84 (nucleotide 251), a mutation at codon 85 (nucleotide 253), and mutations at both, were used to examine the total genomic DNA from each of the 30 isolates, which had been restricted, electrophoresed, and Southern blotted. The probes indicated that 15 of the 28 ciprofloxacin-resistant isolates gave results consistent with a single mutation at codon 84. Four of the 28 ciprofloxacin-resistant strains had results consistent with a mutation at codon 84 and possibly at codon 85. The two ciprofloxacin-sensitive isolates from Tennessee showed homology with the wild-type probe sequence. Five isolates (4, Minnesota; 1, Tennessee) had no homology with any probe. By oligonucleotide probes, ciprofloxacin-resistant MRSA from diverse geographic regions contained similar gyrA mutations at codons 84 or 85 in 19 of 28 ciprofloxacin-resistant isolates.

Base Sequence↗
PubMed1991

Ciprofloxacin resistance in coagulase-positive and -negative staphylococci: role of mutations at serine 84 in the DNA gyrase A protein of Staphylococcus aureus and Staphylococcus epidermidis.

gyrA mutations in quinolone-resistant pathogenic isolates of Staphylococcus spp. have been detected by the direct HinfI digestion of polymerase chain reaction products. Homology among gyrA genes allowed rapid examination of both coagulase-positive and -negative isolates. DNA sequence analysis revealed that ciprofloxacin resistance in Staphylococcus epidermidis is associated with a novel Ser-84----Phe mutation in the DNA gyrase A protein, analogous to Ser-84----Leu changes observed in Staphylococcus aureus.

Anti-Infective Agents↗
PubMed1991

4-Quinolone resistance mutations in the DNA gyrase of Escherichia coli clinical isolates identified by using the polymerase chain reaction.

Seven nalidixic acid-resistant clinical isolates of Escherichia coli were shown to carry resistance mutations in their gyrase A proteins. Six had serine-83 to leucine or tryptophan changes; the seventh had an aspartate-87 to valine substitution. The frequent occurrence of a mutation at serine-83 implies a key role for this residue in quinolone action.

Amino Acid Sequence↗
PubMed1991

Aging and slowing of behavior: consequences for cognition and survival.

It has been over a hundred years since Francis Galton observed that older persons responded more slowly than the young. Since then, slowness of behavior with age has become the most robust phenomenon seen in research on aging. Interest in the slowness of behavior with age, in fact, has been growing in scientific significance because of the relation of speed to cognitive functions, on the one hand, and to physical fitness and electrophysiological measurements on the other. In addition, speed of behavior may be viewed as a reliable marker of aging. This broader context gives rise to the question of how speed of behavior is related to the hierarchical organization of the functions of the central nervous system, a topic neglected by both general and developmental psychologists. Speed of behavior apparently limits certain intellectual components such as fluid intelligence and the important precognitive capacity of attention. Although there may be several speed factors, at least one broad one has pervasive influence on abilities. It appears that the relations among age, speed, and cognitive abilities are not similar to those of other domains of general psychology, and for that reason changes in speed and its role in behavior cannot easily be captured in terms of familiar metaphors of psychology. We must seek new metaphors that can characterize the complexity of aging as a process of transformation and also embrace the apparent contradictions seen in simultaneous increases and decreases in behavioral capacities with age. New research should be considered to take advantage of the robustness of the observed slowness of behavior with age and its widespread correlation with other behavioral and physiological functions. As one of the most reliable psychological markers of aging, it may be used in longitudinal studies to gain further insight into the organization of aging. One of the topics speed of behavior may contribute to is the relation of an energetic concept of aging involving metabolism and capacity for work to an informational or control concept--for example, gaining some insight into the relative contributions of "software" and "hardware" factors in human aging. It appears that in mammals a relatively large nervous system is related to a longer life span. Perhaps for humans, the eventual limiting factor on longevity is the integrity of the nervous system with its fixed long-lived neurons. Future studies of aging may well place greater emphasis on the part the central nervous system plays in aging owing to its role as the integrator of bodily functions.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1990

Relationship of major histocompatibility complex class II genes to inhibitor antibody formation in hemophilia A.

Approximately 14% of transfused hemophiliacs develop an anti-factor VIII inhibitory antibody which specifically neutralizes factor VIII procoagulant activity. In this study an association of the major histocompatibility complex (MHC) with inhibitor antibody formation was evaluated by restriction fragment length polymorphism (RFLP) analysis using BamHI, EcoRI, HindIII, PstI, PvuII and TaqI digested genomic DNA probed with DP beta, DQ alpha, DQ beta and DR beta class II MHC gene probes. The RFLP patterns for 16 non-inhibitor and 11 inhibitor hemophiliac patients were analyzed. These 24 enzyme:probe combinations generated 231 fragments. Fifteen (15) fragments associated with the inhibitor phenotype; odds ratios ranged from 5.1 to 45 and lower bounds of 95% confidence intervals were greater than 1.000 for all 15 fragments. Five (5) fragments associated with non-inhibitors, with odds ratios ranging from 6.4 to 51.7. This report establishes a MHC related genetic basis for the inhibitor phenotype. No statistically significant differences in the distribution of serologically defined HLA-DR phenotypes were observed between the inhibitor and non-inhibitor groups.

Adsorption↗
PubMed1990

Structure and partial amino acid sequence of calf thymus DNA topoisomerase II: comparison with other type II enzymes.

The partial amino acid sequence of p140 calf thymus DNA topoisomerase II was determined by analysis of cyanogen bromide peptides. Five peptides were aligned and shared extensive homology with sequences derived from cDNA clones for the human topoisomerase II isoenzyme forms. Less homology was seen with the Drosophila, yeast and bacterial type II enzymes. Calf and human enzymes shared epitopes allowing isolation of a cDNA clone to human topoisomerase II isoenzyme alpha. Our results indicate that calf thymus p140 topoisomerase II is an active N-terminal proteolytic fragment of the native p180 enzyme and demonstrate that mammalian type II enzymes exhibit close sequence similarity.

Amino Acid Sequence↗