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L F Liu

Publications and source records attributed to L F Liu.

At least 73 records · Page 4Linked to original sources
PubMed1996

DNA rearrangement mediated by inverted repeats.

Inverted repeats of DNA are widespread in the genomes of eukaryotes and prokaryotes and can mediate genome rearrangement. We studied rearrangement mediated by plasmid-borne inverted repeats in Escherichia coli. We show that inverted repeats can mediate an efficient and recA-independent recombination event. Surprisingly, the product of this recombination is not that of simple inversion between the inverted repeats, but almost exclusively an unusual head-to-head dimer with complex DNA rearrangement. Moreover, this recombination is dramatically reduced by increasing the distance separating the repeats. These results can be readily explained by a model involving reciprocal switching of the leading and lagging strands of DNA replication within the inverted repeats, which leads to the formation of a Holliday junction. Reciprocal strand switching during DNA replication might be a common mechanism for genome rearrangement associated with inverted duplication.

Antiporters↗
PubMed1996

Functional analysis of human/chicken transferrin receptor chimeras indicates that the carboxy-terminal region is important for ligand binding.

Chimeric human/chicken transferrin receptors have been constructed using the polymerase chain reaction. Different regions of the 671-residue external domain of the human transferrin receptor were replaced by the corresponding sequences from the chicken transferrin receptor. As chicken transferrin receptors do not bind human transferrin, functional analysis of such chimeric receptors provides an approach to define the ligand-binding site of the human transferrin receptor. Four of 16 chimeric human/chicken transferrin receptors expressed in chick embryo fibroblasts were efficiently transported to the plasma membrane and displayed on the cell surface. Studies of the four chimeric receptors indicated that binding of human transferrin was abolished if the carboxy terminal 192 amino acids of the human transferrin receptor (residues 569-760) were replaced with the corresponding region from the chicken transferrin receptor. Further, a chimeric receptor in which the carboxy-terminal 72 residues were derived from the chicken transferrin receptor exhibited a 16-fold decrease in binding affinity for human transferrin. In contrast, analysis of the other two chimeric receptors showed that 340 amino acids of the human transferrin receptor external domain more proximal to the transmembrane region (residues 151-490) could be replaced with the corresponding region from the chicken transferrin receptor without loss of high-affinity ligand binding. In contrast, two mAbs against the human transferrin receptor external domain, B3/25 and D65.3, that do not compete with transferrin binding, do not bind the chimeric transferrin receptors in which the membrane proximal part is replaced by chicken sequences, while they do bind the two other chimeric transferrin receptors with high affinity. These data indicate that sequence differences in the carboxy-terminal region of human and chicken transferrin receptor external domains are important for the species specificity of transferrin binding and imply that this portion of the human transferrin receptor is critical for ligand binding.

Animals↗
PubMed1996

Sugars act as signal molecules and osmotica to regulate the expression of alpha-amylase genes and metabolic activities in germinating cereal grains.

The molecular mechanisms that initiate and control the metabolic activities of seed germination are largely unknown. Sugars may play important roles in regulating such metabolic activities in addition to providing an essential carbon source for the growth of young seedlings and maintaining turgor pressure for the expansion of tissues during germination. To test this hypothesis, we investigated the physiological role of sugars in the regulation of alpha-amylase gene expression and carbohydrate metabolism in embryo and endosperm of germinating rice seeds. RNA gel blot analysis revealed that in the embryo and aleurone cells, expression of four alpha-amylase genes was differentially regulated by sugars via mechanisms beyond the well-known hormonal control mechanism. In the aleurone cells, expression of these alpha-amylase genes was regulated by gibberellins produced in the embryo and by osmotically active sugars. In the embryo, expression of two alpha-amylase genes and production of gibberellins were transient, and were probably induced by depletion of sugars in the embryo upon imbibition, and suppressed by sugars influx from the endosperm as germination proceeded. The deferential expression of the four alpha-amylase genes in the embryo and aleurone cells was probably due to their markedly different sensitivities to changes in tissue sugar levels. Our study supports a model in which sugars regulate the expression of alpha-amylase genes in a tissue-specific manner: via a feedback control mechanism in the embryo and via an osmotic control mechanism in the aleurone cells. An interactive loop among sugars, gibberellins, and alpha-amylase genes in the germinating cereal grain is proposed.

Carbohydrates↗
PubMed1996

Structure-activity relationships of benzimidazoles and related heterocycles as topoisomerase I poisons.

A series of substituted 2-(4-methoxyphenyl)-1H-benzimidazoles were synthesized and evaluated as inhibitors of topoisomerase I. The presence of a 5-formyl-, 5-(aminocarbonyl)-, or 5-nitro group (i.e., substituents capable of acting as hydrogen bond acceptors) correlated with the potential of select derivatives to inhibit topoisomerase I. In contrast to bi- and terbenzimidazoles, the substituted benzimidazoles that were active as topoisomerase I poisons exhibited minimum or no DNA binding affinity. 5-Nitro-2-(4-methoxyphenyl)-1H-benzimidazole exhibited the highest activity and was significantly more active than the 4-nitro positional isomer. The 5- and 6-nitro derivatives of 2-(4-methoxyphenyl) benzoxazole, 2-(4-methoxyphenyl)benzothiazole, and 2-(4-methoxyphenyl)indole were synthesized and their relative activity as topoisomerase I inhibitors determined. None of these heterocyclic analogues were effective in significantly inhibiting cleavable-complex formation in the presence of DNA and topoisomerase I, suggesting a high degree of structural specificity associated with the interaction of these substituted benzimidazoles with the enzyme or the enzyme-DNA complex. In evaluating their cytotoxicity, these new topoisomerase I poisons also exhibited no significant cross-resistance against cell lines that express camptothecin-resistant topoisomerase I.

Antineoplastic Agents, Phytogenic↗
PubMed1996

Coralyne and related compounds as mammalian topoisomerase I and topoisomerase II poisons.

DNA topoisomerases are nuclear enzymes responsible for modifying the topological state of DNA. The development of agents capable of poisoning topoisomerases has proved to be an attractive approach in the search for novel cancer chemotherapeutics. Coralyne, an antileukemic alkaloid, has appreciable structural similarity to the potent topoisomerase I and II poison, nitidine. Analogues of coralyne were synthesized and evaluated for their activity as topoisomerase I and topoisomerase II poisons. These analogues were also evaluated for cytotoxicity in the human lymphoblast cell line, RPMI 8402, and its camptothecin-resistant variant, CPT-K5. The pharmacological activity of these analogues exhibited a strong dependence on the substitution pattern and the nature of substituents. Several 1-benzylisoquinolines and 3-phenylisoquinolines were also synthesized. These compounds, which incorporate only a portion of the ring structure of coralyne, were evaluated as topoisomerase poisons and for cytotoxicity. These structure-activity studies indicate that the structural rigidity associated with the coralyne ring system may be critical for pharmacological activity. The presence of a 3,4-methylenedioxy substituent on these coralyne analogues was generally associated with enhanced activity as a topoisomerase poison. 5,6-Dihydro-3,4-methylenedioxy-10,11-dimethoxydibenzo[a,g]quinoliz inium chloride was the most potent topoisomerase I poison among the coralyne analogues evaluated, having similar activity to camptothecin. This analogue also possessed exceptional potency as a topoisomerase II poison. Despite the pronounced activity of several of these coralyne derivatives as topoisomerase I poisons, none of these compounds had cytotoxic activity similar to camptothecin. Possible differences in cellular absorption between these coralyne analogs, which possess a quaternary ammonium group, and camptothecin may be responsible for the differences observed in their relative cytotoxicity.

Antineoplastic Agents↗
PubMed1996

Characterizing the DNA binding modes of a topoisomerase I-poisoning terbenzimidazole: evidence for both intercalative and minor groove binding properties.

We have used a broad range of spectroscopic and viscometric techniques to demonstrate that the complexation of a cytotoxic, topoisomerase I-poisoning terbenzimidazole (5PTB) with the poly(dA).poly(dT) duplex exhibits properties characteristic of both intercalation and minor groove binding. Our results reveal the following features: (i) Optical melting profiles reveal that 5PTB binding enhances the thermal stability of the poly(dA).poly(dT) duplex; (ii) Fluorescence-detected 5PTB binding to the poly(dA).poly(dT) duplex reveals four apparent "site sizes," ranging from 1 to 13 base pairs (bp) per bound drug; (iii) Flow linear dichroism data suggest conformational heterogeneity among the poly(dA).poly(dT)-bound 5PTB molecules, with substantial contributions from drug molecules bound in the minor groove; (iv) Fluorescence resonance energy transfer data reveal properties characteristic of a significant contribution from an intercalative mode of binding; (v) Viscometric, fluorescence quenching, and netropsin competition data are consistent with 5PTB binding to poly(dA).poly(dT) by "mixed" modes, which are operationally defined as single or multiple binding populations that individually and/or collectively express both intercalative and minor groove binding properties. We comment on a potential correlation between drugs that exhibit such "mixed" mode binding motifs and those that express antineoplastic activity through inhibition of topoisomerase I.

Benzimidazoles↗
PubMed1995

Synthesis and evaluation of terbenzimidazoles as topoisomerase I inhibitors.

The synthesis and pharmacological activity of a series of terbenzimidazoles are described. The ability of these derivatives to induce DNA cleavage in the presence of topoisomerase I was evaluated in vitro. These analogs were also assayed for their cytotoxicity in RPMI 8402 cells and the camptothecin-resistant CPT-K5 cells. In addition the potential for these compounds to serve as substrates for MDR1 was also determined. Several terbenzimidazoles exhibited similar cytotoxicity against variants of human tumor cells that either overexpress MDR1 or are camptothecin-resistant.

Animals↗
PubMed1995

Specific stimulation of recA-independent plasmid recombination by a DNA sequence at a distance.

Recombination between directly repeated DNA sequences can occur via both recA-dependent and recA-independent mechanisms. They are differentially affected by the length of the repeat and the distance separating the repeats, respectively. Interestingly, a 623 base-pairs long DNA sequence of the plasmid pBR322 was found to stimulate specifically recA-independent recombination between tandem repeats. Analysis of this stimulating sequence has revealed the following features. (1). It is cis-acting. (2). No specific region of it appears to be essential for the effect. Moreover, a neutral sequence of comparable size was able to substitute for the sequence in influencing recombination. (3). The sequence affects recombination between tandem repeats within the tetA but not the bla gene of pBR322. (4). The sequence exerts its effect in a position-dependent manner. (5). It changes not only the frequency but also the products of recombination. Our results provide an example that recA-independent recombination can be influenced by DNA sequences at a distance.

Antiporters↗
PubMed1995

Overexpression of RNase H partially complements the growth defect of an Escherichia coli delta topA mutant: R-loop formation is a major problem in the absence of DNA topoisomerase I.

Previous biochemical studies have suggested a role for bacterial DNA topoisomerase (TOPO) I in the suppression of R-loop formation during transcription. In this report, we present several pieces of genetic evidence to support a model in which R-loop formation is dynamically regulated during transcription by activities of multiple DNA TOPOs and RNase H. In addition, our results suggest that events leading to the serious growth problems in the absence of DNA TOPO I are linked to R-loop formation. We show that the overexpression of RNase H, an enzyme that degrades the RNA moiety of an R loop, can partially compensate for the absence of DNA TOPO I. We also note that a defect in DNA gyrase can correct several phenotypes associated with a mutation in the rnhA gene, which encodes the major RNase H activity. In addition, we found that a combination of topA and rnhA mutations is lethal.

Cold Temperature↗
PubMed1995

Cell cycle-specific and transcription-related phosphorylation of mammalian topoisomerase I.

Eukaryotic DNA topoisomerase I has been recently shown to be associated with the transcriptional machinery and has also been implicated to function in DNA replication and perhaps other DNA transactions. We have identified several differentially phosphorylated forms of mammalian topoisomerase I as electrophoretic variants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These differently phosphorylated forms cleave chromosomal DNA in cells and also relax supercoiled DNA with about equal activity, suggesting that they primarily function other than to activate catalysis. One of the phosphorylated forms is specifically present during mitosis. Upon transition from mitosis into G1 phase, two forms differing in phosphorylation state appear and persist throughout the remainder of interphase. When cells are incubated in a pellet, one of the interphase phosphorylated forms disappears coincidentally with an increase in the abundance of the other; if the cell pellet is disrupted and the cells are reincubated in suspension, the forms rapidly shift back to their original abundance levels. Finally, a shift in relative abundance of the differently phosphorylated interphase forms is observed when transcription is inhibited. These results suggest that dynamic phosphorylation and dephosphorylation regulate topoisomerase I during RNA transcription and cell cycle progression.

3T3 Cells↗
PubMed1994

Modification of glutathione S-transferase 3-3 mutants with 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone. Identification of the C-terminal tryptic fragment as part of the H-site and evidence that 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone is not specific for cysteine labelling.

A triple mutant of rat liver glutathione S-transferase 3-3 that has all three cysteine residues replaced with serine (CallS) and a quadruple mutant with a Tyr-115 to phenylalanine substitution on CallS (CallSY115F) were reacted with 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone (GS-1,4-TCBQ). The modified proteins were analysed on a triple-quadrupole mass spectrometer equipped with an electrospray ionization source. At an enzyme: GS-1,4-TCBQ ratio of 1:10, the enzymes were modified at multiple sites. Covalent attachment of a single inhibitor on to the protein was achieved by lowering the enzyme: GS-1,4-TCBQ ratio to 1:1. Results from m.s. analyses suggest that the inhibitor on the CallSY115F mutant exists as a glutathionyl dichlorobenzoquinone derivative. The modifiers of the CallS mutants are glutathionyl monochlorobenzoquinone derivatives. Therefore, GS-1,4-TCBQ reacts at a single site on CallSY115F, but probably cross-links two regions on wild-type and CallS mutant. To confirm our observation, CallS was modified with 1-chloro2,4-dinitrobenzene, which specifically labels Tyr-115, before reacting with GS-1,4-TCBQ. The inhibitor formed a glutathionyl dichlorobenzoquinone adduct on the dinitrophenyl-CallS mutant. In addition, the benzoquinone derivative on the protein can be partially removed by 1-chloro-2,4-dinitrobenzene. Peptide mapping and sequencing analysis of the GS-1,4-TCBQ-modified CallS mutant revealed that the C-terminal 16-amino-acid fragment is labelled. Molecular modelling suggests the C(5) and C(6) on the benzoquinone ring of the inhibitor interact with the oxygen atoms of Tyr-115 and Ser-209 respectively.

Amino Acid Sequence↗
PubMed1994

Synthesis and pharmacological evaluation of isoindolo[1,2-b]quinazolinone and isoindolo[2,1-a]benzimidazole derivatives related to the antitumor agent batracylin.

The synthesis and pharmacological activity of isoindolo[1,2-b]quinazolin-12(10H)-ones and isoindolo[2,1-a]benzimidazoles related to batracylin are described. The acute toxicity of batracyclin has been associated with the formation of its N-acetyl metabolite which is a potent inducer of unscheduled DNA synthesis in rat hepatocytes. The desamino derivative and the 8-aza analog of batracylin retained the ability to inhibit topoisomerase II but did not induce unscheduled DNA synthesis. While less active than batracylin, these analogs were cytotoxic to CCRF CEM leukemia cells. The isoindolo[2,1-a]benzimidazole derivatives were inactive as topoisomerase II inhibitors and, in general, failed to exhibit comparable antitumor activity or to induce unscheduled DNA synthesis.

Animals↗
PubMed1994

Hypernegative supercoiling of the DNA template during transcription elongation in vitro.

Supercoiled plasmid DNAs with negative superhelicity several times higher than normal have been isolated from Escherichia coli topA mutants. The formation of these hypernegatively supercoiled plasmid DNAs is apparently induced by transcription. We show that hypernegatively supercoiled plasmid DNAs isolated from topA mutants contain R-loop(s). To study the mechanism of formation of hypernegatively supercoiled plasmid DNA, we have been able to reproduce hypernegatively supercoiled DNA in vitro using purified RNA polymerase and DNA gyrase. The formation of hypernegatively supercoiled plasmid DNA template in vitro is shown to require transcription elongation and is tightly linked to R-loop formation. We propose that one of the roles of topoisomerase I is to suppress R-loop formation during transcription elongation.

DNA Topoisomerases, Type I↗
PubMed1994

recA-independent and recA-dependent intramolecular plasmid recombination. Differential homology requirement and distance effect.

We show that recombination between directly repeated sequences in plasmids occurs via both recA-independent and recA-dependent mechanisms in Escherichia coli. They are differentially affected by two factors, the distance separating the homologies and the size of the homology. Recombination between tandem duplications up to 300 base-pairs shows virtually no recA dependence. Increasing the size of the duplications beyond 300 base-pairs gradually increases the recA dependence. Furthermore, insertion of a sizable DNA sequence in between the duplications, substantially increases the recA dependence. We conclude that increasing the distance separating the homologous regions preferentially inhibits the recA-independent recombination. On the other hand, shortening of the homology preferentially inhibits recA-dependent recombination. Consequently, recombination between short tandem duplications is totally recA-independent.

Base Sequence↗