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D S Samuels

Publications and source records attributed to D S Samuels.

32 records · Page 2Linked to original sources
PubMed1994

Phosphorylation of DNA topoisomerase I is increased during the response of mammalian cells to mitogenic stimuli.

DNA topoisomerase I is phosphorylated after mitogenic stimulation of 3T3-L1 mouse fibroblasts by 12-O-tetradecanoylphorbol 13-acetate (TPA), a phorbol ester tumor promoter. In vivo labeling with [32P]orthophosphate and immunoprecipitation with an anti-DNA topoisomerase I antibody has demonstrated an increase in the phosphorylation of DNA topoisomerase I in Swiss/3T3 mouse fibroblasts treated with epidermal growth factor (EGF) and H35 rat hepatoma cells treated with insulin. The only phosphorylated form of DNA topoisomerase I observed was the 100-kDa protein Digestion of DNA topoisomerase I with trypsin revealed two phosphopeptides. In addition, VT-1, a non-responsive genetic variant of 3T3-L1, and the DNA topoisomerase I inhibitor camptothecin were used to further study TPA-induced DNA topoisomerase I phosphorylation. Our results indicate that the phosphorylation of DNA topoisomerase I may be an ubiquitous response of cultured mammalian cells to mitogenic agents, even in the absence of DNA replication.

3T3 Cells↗
PubMed1994

The predominant form of mammalian DNA topoisomerase I in vivo has a molecular mass of 100 kDa.

DNA topoisomerase I was purified to apparent homogeneity from human HeLa cells as a single polypeptide with a molecular mass of 100 kDa, as assayed by both gel filtration column chromatography and SDS-polyacrylamide gel electrophoresis. No smaller forms of the enzyme were detected in the purified fraction. Therefore, smaller forms, which have been observed by other investigators, are likely to be the result of proteolysis during isolation and are not relevant to the in vivo activity of DNA topoisomerase I.

Artifacts↗
PubMed1994

gyrB mutations in coumermycin A1-resistant Borrelia burgdorferi.

We have isolated and characterized mutants of Borrelia burgdorferi that are resistant to the antibiotic coumermycin A1, which targets the B subunit of DNA gyrase. Mutants had either 100- or 300-fold higher resistance to coumermycin A1 than wild-type B. burgdorferi. In each case, a single point mutation in the gyrB gene converted Arg-133 to Gly or Ile. Mutations in the homologous Arg residue of Escherichia coli DNA gyrase are also associated with resistance to coumarin antimicrobial agents.

Amino Acid Sequence↗
PubMed1994

Analysis of the distribution and molecular heterogeneity of the ospD gene among the Lyme disease spirochetes: evidence for lateral gene exchange.

Analysis of the ospD gene has revealed that this gene is not universal among Lyme disease spirochete isolates. The gene was found to be carried by 90, 50, and 24% of the Borrelia garinii, B. afzelii, and B. burgdorferi isolates tested. Size variability in the ospD-encoding plasmid was also observed. Sequence analysis has demonstrated the presence of various numbers of a 17-bp repeated sequence in the upstream control (promoter) region of the gene. In addition, a region within the coding sequence where various insertions, deletions, and direct repeats occur was identified. ospD gene sequences from 31 different isolates were determined and utilized in pairwise sequence comparisons and construction of a gene tree. These analyses suggest that the ospD gene was the target of several recombinational events and that the gene was recently acquired by Lyme disease spirochetes and laterally transferred between species.

Amino Acid Sequence↗
PubMed1994

Genetic transformation of the Lyme disease agent Borrelia burgdorferi with coumarin-resistant gyrB.

No useful method to genetically manipulate Borrelia burgdorferi, the causative agent of Lyme disease, has been developed previously. We have used resistance to the coumarin antibiotic coumermycin A1, an inhibitor of DNA gyrase, as a genetic marker to monitor the transformation of B. burgdorferi by electroporation. Introduction of site-directed mutations into the gyrB gene demonstrated that transformation was successful, provided evidence that homologous recombination occurs on the chromosome, and established that mutations at Arg-133 of DNA gyrase B confer coumermycin A1 resistance in B. burgdorferi. The coumermycin A1-resistant gyrB marker and genetic transformation can now be applied toward dissecting the physiology and pathogenesis of the Lyme disease agent on a molecular genetic level.

Aminocoumarins↗
PubMed1993

Variation in the size of the ospA-containing linear plasmid, but not the linear chromosome, among the three Borrelia species associated with Lyme disease.

The aetiological agents of Lyme disease form a phylogenetically heterogeneous group, composed of three species, Borrelia burgdorferi, Borrelia garinii, and group VS461. We have compared the sizes of the linear plasmid that carries the genes encoding the major outer-surface proteins OspA and OspB as well as the size and structure of the chromosome among the Lyme disease spirochaetes. We have found differences in the sizes of the ospA-containing plasmids, but not the linear chromosomes among the three species. The ospA-containing plasmid size of 50 kb in B. burgdorferi isolates is significantly smaller than the size of 55 kb in B. garinii isolates and 56 kb in group VS461 isolates. The chromosome was found to be linear in all three Borrelia species, but not significantly different in size.

Antigens, Surface↗
PubMed1993

Coumermycin A1 inhibits growth and induces relaxation of supercoiled plasmids in Borrelia burgdorferi, the Lyme disease agent.

Coumermycin A1 is an inhibitor of DNA gyrase, an enzyme that catalyzes supercoiling of DNA and is required for bacterial DNA replication. We have investigated the activity of this coumarin antibiotic on Borrelia burgdorferi, a spirochete and the causative agent of Lyme disease. B. burgdorferi was more susceptible than many other eubacteria to coumermycin as well as novobiocin, another coumarin antibiotic; this contrasted with its relative resistance to the DNA gyrase inhibitors nalidixic acid, oxolinic acid, and ciprofloxacin. Coumermycin at 0.2 micrograms/ml inhibited the growth of B. burgdorferi B31 in BSK II medium. A 100-fold-lower concentration induced the relaxation of two negatively supercoiled circular plasmids within 2 h. Plasmid supercoiling was restored within 2 h of removal of coumermycin. These results suggest that B. burgdorferi has a DNA gyrase and that this enzyme's activity is required for growth. Furthermore, structural analogs of coumermycin may be considered as treatments for Lyme disease.

Aminocoumarins↗
PubMed1993

Transcriptional analyses and mapping of the ospC gene in Lyme disease spirochetes.

In Lyme disease spirochetes, the ospC gene encodes a 22.7-kDa protein referred to as either the pC or the OspC protein. Using a variety of electrophoretic approaches followed by Southern blotting and probing with oligonucleotide probes, we mapped the ospC gene to a circular 26-kb plasmid. The ospC gene represents the first gene to be mapped to a circular plasmid in Lyme disease spirochetes. The occurrence of this gene in isolates belonging to each of the three Lyme disease-associated species, Borrelia burgdorferi, Borrelia garinii, and the VS461 group, was evaluated. The ospC gene was found to occur in all 21 isolates tested from each of the three species. Differential hybridization with a series of ospC probes in both Northern (RNA) and Southern blot analyses demonstrated that there is sequence variability in the ospC gene among isolates. While the gene was found to be present in all isolates, not all actively transcribed the gene. Transcriptional start site analyses suggest that the gene may be under the control of multiple promoters that are highly similar in nucleotide sequence.

Antigens, Bacterial↗
PubMed1993

Identification of a protein in several Borrelia species which is related to OspC of the Lyme disease spirochetes.

Using oligonucleotide probes which have previously been shown to be specific for the ospC gene found in the Lyme disease spirochete species Borrelia burgdorferi, B. garinii, and group VS461, we detected an ospC homolog in other Borrelia species including B. coriaceae, B. hermsii, B. anserina, B. turicatae, and B. parkeri. In contrast to the Lyme disease spirochetes, which carry the ospC gene on a 26-kb circular plasmid, we mapped the gene in other Borrelia species to linear plasmids which varied in size among the isolates tested. Some isolates carry multiple copies of the gene residing on linear plasmids of different sizes. The analyses conducted here also demonstrate that these Borrelia species contain a linear chromosome. Northern (RNA) blot analyses demonstrated that the gene is transcriptionally expressed in all species examined. High levels of transcriptional expression were observed in some B. hermsii isolates. Transcriptional start site analyses revealed that the length of the untranslated leader sequence was identical to that observed in the Lyme disease spirochete species. By Western blotting (immunoblotting) with antiserum (polyclonal) raised against the OspC protein of B. burgdorferi, we detected an immunoreactive protein of the same molecular weight as the OspC found in Lyme disease spirochete species. The results presented here demonstrate the presence of a protein that is genetically and antigenically related to OspC which is expressed in all species of the genus Borrelia tested.

Antigens, Bacterial↗
PubMed1992

DNA topoisomerase I phosphorylation in murine fibroblasts treated with 12-O-tetradecanoylphorbol-13-acetate and in vitro by protein kinase.

The phosphorylation of DNA topoisomerase I in quiescent murine 3T3-L1 fibroblasts treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was characterized by in vivo labeling with [32P] orthophosphate and immunoprecipitation with a scleroderma anti-DNA topoisomerase I autoantibody. DNA topoisomerase I phosphorylation was stimulated 4-fold by 2 h of TPA treatment (TPA at 100 ng/ml maximally enhanced phosphorylation). Purified DNA topoisomerase I was phosphorylated in vitro in a Ca2+ and phospholipid-dependent fashion by types I, II, and III protein kinase C. The phosphorylation reaction was stimulated by TPA and had an apparent K(m) of 0.4 microM. DNA topoisomerase I was phosphorylated in vivo and in vitro predominantly at serine. The major tryptic phosphopeptides from DNA topoisomerase I in TPA-treated fibroblasts and phosphorylated by protein kinase C comigrated in thin-layer electrophoresis. The half-life of incorporated phosphate on DNA topoisomerase I was 40 min in both TPA-treated and control cells. These results suggest that phosphorylation is a mechanism for activating DNA topoisomerase I in fibroblasts treated with TPA and that protein kinase C functions in the phosphorylation.

3T3 Cells↗
PubMed1991

Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 51, a residue crucial to its trans-activation function.

The vitamin D receptor (VDR) is known to be a phosphoprotein and inspection of the deduced amino acid sequence of human VDR (hVDR) reveals the conservation of three potential sites of phosphorylation by protein kinase C (PKC)--namely, Ser-51, Ser-119, and Ser-125. Immunoprecipitated extracts derived from a rat osteoblast-like osteosarcoma cell line that contains the VDR in high copy number were incubated with the alpha, beta, and gamma isozymes of PKC, and VDR proved to be an effective substrate for PKC-beta, in vitro. When hVDR cDNAs containing single, double, and triple mutations of Ser-51, Ser-119, and Ser-125 were expressed in CV-1 monkey kidney cells, immunoprecipitated and phosphorylated by PKC-beta, in vitro, the mutation of Ser-51 selectively abolished phosphorylation. Furthermore, when transfected CV-1 cells were treated with phorbol 12-myristate 13-acetate, a PKC activator, phosphorylation of wild-type hVDR was enhanced, whereas that of the Ser-51 mutant hVDR was unaffected. Therefore, Ser-51 is the site of hVDR phosphorylation by PKC, both in vitro and in vivo. To evaluate the functional role of Ser-51 and its potential phosphorylation, hVDR-mediated transcription was tested using cotransfection with expression plasmids and a reporter gene that contained a vitamin D response element. Mutation of Ser-51 markedly inhibited transcriptional activation by the vitamin D hormone, suggesting that phosphorylation of Ser-51 by PKC could play a significant role in vitamin D-dependent transcriptional activation. Therefore, the present results link the PKC signal transduction pathway of growth regulation and tumor promotion to the phosphorylation and function of VDR.

Amino Acid Sequence↗
PubMed1989

Protein kinase C phosphorylates DNA topoisomerase I.

The induction of mammalian cell proliferation requires the expression of a specific set of genes. Tumor promoters stimulate cell growth by activating the Ca2+ and phospholipid-dependent protein kinase, protein kinase C (PKC). DNA topoisomerase I, a nuclear enzyme involved in transcription, was phosphorylated by activated PKC in vitro. Phosphorylation by PKC stimulated the DNA relaxation activity of topoisomerase I two- to three-fold. Therefore, DNA topoisomerase I is a substrate for PKC-mediated activation by phosphorylation and may serve as a nuclear target of mitogenic signals generated by tumor promoters in vivo.

DNA Topoisomerases, Type I↗
PubMed1986

Inhibition of topoisomerase I by antibodies in sera from scleroderma patients.

Purified type I topoisomerase from calf thymus as well as nuclear and cytoplasmic extracts from EGF-stimulated human and mouse fibroblasts in cell culture efficiently convert supercoiled plasmid DNA to the relaxed form. The purified IgG fraction from the sera of Japanese patients with the rheumatic disease scleroderma were shown to inhibit this relaxation activity. Thus, these patients likely produce autoantibodies to topoisomerase I. In addition, the human, bovine and murine enzymes share antigenic determinants recognized by the antisera.

Animals↗