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At least 433 records · Page 24Linked to original sources
PubMed2004

The role of polyadenylation signal secondary structures on the resistance of plasmid vectors to nucleases.

BACKGROUND: Nuclease degradation of plasmid DNA (pDNA) vectors after delivery and during trafficking to the nucleus is a barrier to gene expression. This barrier may be circumvented by shielding the pDNA from the nuclease-rich cell environment with adjuvants or by using nuclease inhibitors. A different alternative that is explored in this work is to make pDNA vectors more nuclease-resistant a priori. METHODS AND RESULTS: The hypothesis that a significant part of nuclease attack is directed towards certain labile sequences in a pDNA model (pVAX1/lacZ) was first tested. Homopurine-rich tracts in the bovine growth hormone polyadenylation signal (BGH poly A) were identified as labile sequences using S1 nuclease as a probe. Two pDNA variants were then created by replacing the BGH poly A region with the SV40 or a synthetic poly A signal. A study of plasmid degradation in eukaryotic cell lysates and mice plasma showed that the half-life of the supercoiled isoforms of the new vectors was always higher when compared with the control plasmid. An in vitro assay of the reporter beta-galactosidase in transfected CHO cells further showed that gene expression with the new pDNA variants was not affected negatively by the plasmid modifications. CONCLUSIONS: The replacement of labile sequences in plasmid DNA vectors improves resistance towards nuclease attack as shown by the increased half-lives of supercoiled plasmid isoforms incubated with endo/lysosomal, cytoplasmatic and blood plasma enzymes.

Animals↗
PubMed2006

The role of a HSV thymidine kinase stimulating substance, scopadulciol, in improving the efficacy of cancer gene therapy.

BACKGROUND: The most extensively investigated strategy of suicide gene therapy for treatment of cancer is the transfer of the herpes simplex virus thymidine kinase (HSV-TK) gene followed by administration of antiviral prodrugs such as acyclovir (ACV) and ganciclovir (GCV). The choice of the agent that can stimulate HSV-TK enzymatic activity is one of the determinants of the usefulness of this strategy. Previously, we found that a diterpenoid, scopadulciol (SDC), produced a significant increase in the active metabolite of ACV. This suggests that SDC may play a role in the HSV-TK/prodrug administration system. METHODS: The anticancer effect of SDC was evaluated in HSV-TK-expressing (TK+) cancer cells and nude mice bearing TK+ tumors. In vitro and in vivo enzyme assays were performed using TK+ cells and tumors. The phosphorylation of ACV monophosphate (ACV-MP) was measured in TK- cell lysates. The pharmacokinetics of prodrugs was evaluated by calculating area-under-the-concentration-time-curve values. RESULTS: SDC stimulated HSV-TK activity in TK+ cells and tumors, and increased GCV-TP levels, while no effect of SDC was observed on the phosphorylation of ACV-MP to ACV-TP by cellular kinases. The SDC/prodrug combination altered the pharmacokinetics of the prodrugs. In accord with these findings, SDC enhanced significantly the cell-killing activity of prodrugs. The bystander effect was also significantly augmented by the combined treatment of ACV/GCV and SDC. CONCLUSIONS: SDC was shown to be effective in the HSV-TK/prodrug administration system and improved the efficiency of the bystander effect of ACV and GCV. The findings will be considerably valuable with respect to the use of GCV in lower doses and less toxic ACV. This novel strategy of drug combination could provide benefit to HSV-TK/prodrug gene therapy.

Abietanes↗
PubMed1988

The chemical carcinogen, chloroacetaldehyde, modifies a specific site within the regulatory sequence of human cytomegalovirus major immediate early gene in vivo.

The reaction of chemical carcinogens with DNA is well documented, but whether this interaction occurs at specific sites in chromatin is unknown. We have examined in vivo the reaction of a known carcinogen, chloroacetaldehyde, with the active and inactive major immediate early gene of human cytomegalovirus. We found that during active transcription of the gene, this chemical carcinogen reacts with a unique DNA site in the 5' flanking sequence of the major immediate early gene. However, no reaction was detected in infected nonpermissive cells in which the gene was inactive. The chloroacetaldehyde-reactive site is located at -836 +/- 10 bp from the mRNA cap site in the part of the regulatory region that can both negatively and positively affect promoter activity [Nelson et al., Mol Cell Biol 7:4125-4129, 1987]. These results suggest, at least in the case of chloroacetaldehyde, the possibility that the molecular mechanism of chemical carcinogenesis involves a chemical reaction at specific sites in chromatin within the sequences responsible for regulation of gene expression. Such carcinogen-DNA interaction occurs as a consequence of a non-B DNA structure that contains unpaired DNA bases existing at specific sites in chromatin.

Acetaldehyde↗
PubMed1997

Inhibition of the topoisomerase II-DNA cleavable complex by the ortho-quinone derivative of the antitumor drug etoposide (VP-16).

Etoposide (VP-16) is a widely used anticancer drug whose toxicity involves poisoning of topoisomerase II. VP-16 undergoes enzymatic oxido-reductive transformations in cells, resulting in the formation of the ortho-quinone derivative (VPQ) as a major product. The actions of VP-16 and VPQ on purified human topoisomerase II have been compared. Both the parent drug and VPQ are very efficient at trapping the topoisomerase II-DNA cleavable complex, suggesting that methoxy groups on the E-ring are not a prerequisite for activity. Our data also imply that VPQ has more effect than VP-16 on the breakage-reunion equilibrium of topoisomerase II and DNA. The stronger inhibition of the religation of the second strand observed with VPQ suggests it interacts asymmetrically with the two homodimers of topoisomerase II bound to DNA.

DNA Topoisomerases, Type II↗
PubMed1995

A comparison of active site binding of 4-quinolones and novel flavone gyrase inhibitors to DNA gyrase.

The activity of 4-quinolone antibacterials at the enzyme target level is based on the well known and reported observations that 4-quinolone antibacterials target the Gyr A subunit of the DNA gyrase holoenzyme, inhibiting supercoiling while facilitating the "cleavable complex". Such inhibition can be observed by running the in vitro DNA gyrase supercoiling inhibition assay or the "cleavable complex" DNA gyrase assay. Although potency of the gyrase inhibitor is dependent on many factors including permeability and pharmacokinetics, the inherent potency of a gyrase inhibitor lies in its activity against the target enzyme. We have examined the binding activity of novel flavones [Bioorganic & Med. Chem. Letters 3:225-230, 1993] to Escherichia coli DNA gyrase and have found differences in binding consistent with inhibition of DNA gyrase supercoiling and ability to facilitate the cleavable complex, but of different rank order. [3H]norfloxacin was used in vitro competition studies with test compounds, pBR322 and E. coli DNA gyrase. Binding affinity results indicate the rank order of greatest to weakest binding (ability to compete with [3H]norfloxacin) of test compounds: Levofloxacin = ciprofloxacin > ofloxacin > norfloxacin > flavone compounds (including ellagic acid, quercetin, and compounds 5a through 5n [Bioorganic & Med. Chem. Letters 3:225-230, 1993]). Such differences in binding ability of the 4-quinolones and flavones to the ternary complex of DNA.DNA gyrase.drug, as compared to the catalytic inhibition and "cleavable complex" data, suggests a more complex binding of flavones than the previously hypothesized models for 4-quinolone binding.

Anti-Infective Agents↗
PubMed1990

The DNA sequence and structural organization of the GC2 plasmid from the red alga Gracilaria chilensis.

The complete DNA sequence of the circular GC2 plasmid from the red alga Gracilaria chilensis was obtained. It contains 3827 bp and has a base composition of 75% A + T nucleotides. The sequence revealed that GC2 has two inverted repeats, each of 290 nucleotides, that border four long direct tandem repeats of 216 nucleotides. five short, direct, tandem repeats of 21-22 nucleotides were also found in the plasmid. The plasmid sequence has one major open reading frame that could encode a 411 amino acid polypeptide. Finally, the GC2 plasmid is transcriptionally active.

Amino Acid Sequence↗
PubMed1979

Transposition of DNA inserted into deletions of the Tn5 kanamycin resistance element.

Tn5-trp hybrid transposons have been constructed by insertion of a trpPOED Hind III fragment into an in vivo Tn5 internal deletion mutant or by substitution of trp for the internal Tn5 Hind III fragment. These hybrids are called, respectively, Tn409 and Tn410. Both Tn409 and Tn410 will transpose into lambda in the presence of a complementing Tn5 element. In the absence of a wild Tn5, lysogens carrying R1162::Tn409 and R1162::Tn410 plasmids will yield lambdatrp phages at less than six per cent of the complemented frequency. This reduction indicates that Tn409 and Tn410 lack a diffusible transposition function provided by wild Tn5 elements. However, the formation of lambdatrp phages without complementation is real. Most of these transducing particles contain Tn409 and Tn410 still linked to the carrier R1162 plasmid. This observation suggests that uncomplemented Tn409 and Tn410 elements mediate the formation of lambda-transposon-plasmid cointegrate structures. Thus, the missing transposition function may be involved in resolving these cointegrate structures to the final lambda::Tn409 or lambda::Tn410 product.

Chromosome Mapping↗
PubMed1989

The influence of the herpes simplex virus-1 DNA template environment on the regulation of gene expression.

To determine the role of the HSV-1 genome structure and environment on the regulation of gene expression, we constructed recombinant viruses containing a heterologous gene inserted into either the immediate early ICPO or late glycoprotein C (gC) genes of HSV-1. The heterologous gene consisted of the SV40 early promoter (without enhancer sequences) linked to the coding sequences for the bacterial chloramphenicol acetyl transferase (CAT). The expression of CAT was examined in Vero cells infected with either virus (named ICP0-CAT and Sph 6). For both recombinants, expression of CAT was not dependent upon prior viral protein synthesis. The kinetics of expression of CAT-specific mRNA resembled that of the HSV-1 genes into which CAT was inserted. Primer extension analysis revealed that the SV40 promoter is recognized and used when placed in cis in two different HSV-1 genome locations, and Northern hybridization experiments confirmed that the heterologous gene was expressed in the absence of prior viral protein synthesis. Therefore, this gene was not regulated as strictly as an HSV-1 gene, but was influenced by the environment into which it was placed, presumably by factors that are present when the normal viral gene is on.

Base Sequence↗
PubMed1994

Purification and cloning of piscicolin 61, a bacteriocin from Carnobacterium piscicola LV61.

Piscicolin 61, a bacteriocin produced by Carnobacterium piscicola LV61, inhibits the growth of strains of Carnobacterium, Lactobacillus, and Enterococcus. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential hydrophobic interaction and reversed-phase chromatography. The N-terminal amino acid sequence of piscicolin 61 was determined by Edman degradation. The plasmid-located structural gene encoding piscicolin (psc61) was cloned and sequenced. It encoded a primary translation product of 71 amino acid residues, which is cleaved between amino acid residues 18 and 19 to yield the active bacteriocin. The calculated M(r) from the deduced protein sequence, 5052.6, agreed with that obtained by mass spectrometry. Piscicolin 61 did not show any sequence similarities to other known bacteriocins. However, the leader sequence resembled those of the pediocin-like bacteriocins. Piscicolin 61 may be able to form amphiphilic helices and may thus act on the membrane of sensitive cells.

Amino Acid Sequence↗
PubMed1997

Transposon-like structure of a new plasmid-encoded restriction-modification system in Rhizobium leguminosarum VF39SM.

Total DNA isolated from Rhizobium leguminosarum VF39SM cells is resistant to cleavage by the restriction endonuclease PstI. Plasmid curing and transfer studies localized this phenotype to pRleVF39b, the second smallest of six plasmids found in this bacterium. In vitro selection for vector modification was employed to isolate a presumptive methylase gene (M.Rle39BI) from a plasmid gene library. Total and plasmid DNAs isolated from E. coli containing M.RleBI were resistant to digestion by PstI. Sequence data suggested that a putative restriction endonuclease (R.Rle39BI) was also encoded on the same fragment. The two genes were flanked by identical copies of a putative insertion sequence, which was also present in several copies elsewhere in the VF39SM genome. The presence of this element in other strains examined suggested that this element is indeed an insertion sequence. The differences in G/C content between the DNA coding for the R/M system and that of the IS element suggest that this DNA region may have been acquired by horizontal transfer.

Amino Acid Sequence↗
PubMed1992

Activation of recA protein. The open helix model for LexA cleavage.

RecA protein is induced by the binding of DNA and ATP to become active in the hydrolysis of ATP and the cleavage of repressors. These reactions appear to depend on the structural state of the protein polymerized along the DNA, i.e. a helical coat of six RecA per turn of 95 to 100 A pitch. In support of this model of the active conformation, it was shown that high concentrations of salt also induce this helical polymerized state as well as the enzymatic activities. Here, we describe that, in vitro and with the non-hydrolyzable analogue ATP gamma S, RNA and heparin can also induce both the structural transition and the enzymatic activation of RecA to LexA cleavage in accordance with the model. RNA and heparin do not support the reaction in the presence of ATP, and they do not induce the hydrolysis of ATP either, suggesting that, in contrast to ATP gamma S, the nucleotide is not bound stably enough, and that the combined affinities of polynucleotide and ATP actually modulate the discrimination of RecA for the various possible inducers in vivo.

Adenosine Triphosphate↗
PubMed1995

Agrocins.

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Adenine Nucleotides↗
PubMed2005

Resveratrol-Cu(II) induced DNA breakage in human peripheral lymphocytes: implications for anticancer properties.

Resveratrol (3,4',5-trihydroxy stilbene), a plant derived polyphenol found in mulberries, grapes and red wine is considered to possess chemopreventive properties against cancer. It is recognized as a naturally occurring antioxidant but also catalyzes oxidative DNA degradation in vitro in the presence of transition metal ions such as copper. Using a cellular system of lymphocytes isolated from human peripheral blood and Comet assay, we have confirmed that resveratrol-Cu(II) system is indeed capable of causing DNA degradation in cells such as lymphocytes. Also, trans-stilbene, which does not have any hydroxyl groups, is inactive in the lymphocyte system. Pre-incubation of lymphocytes with resveratrol indicates that it is capable of either traversing the cell membrane or binding to it. Our results are in partial support of our hypothesis that anticancer properties of various plant derived polyphenols may involve mobilization of endogenous copper and the consequent prooxidant action.

Antineoplastic Agents↗
PubMed2005

Study on the interaction of new water-soluble porphyrin with DNA.

A porphyrin meso-tetrakis{[4-(1-pyridyl)propoxy]phenyl}porphyrin (TPyPP) and its Ni complex (TPyPP(Ni)) have been synthesized and characterized by 1H NMR, UV-vis spectra. The interaction of two porphyrins with calf thymus-DNA (CT-DNA) has been explored by UV-vis, fluorescence and circular dichroic spectroscopy and viscosity measurements. The results suggest that these porphyrins can bind to DNA by the same binding mode. TPyPP outside binds by self-stack with DNA both at low drug load r (=[porphyrin]/[DNA]) and high drug load. Though TPyPP(Ni) has center metal nickel, binding mode with DNA has little difference compared with TPyPP, dominating out-binding mode with different direction along DNA. The binding constants of the TPyPP and TPyPP(Ni) to DNA were 4.65 x 10(5) M(-1) and 3.2 x 10(5) M(-1), respectively. A colored precipitate was found after time in two porphyrin's viscosity measurement. The reasonable interpretation is the porphyrins with alkyl connected N-position of pyridine can strongly interact with the anionic phosphates of DNA and lead to hydrophobic complex.

Animals↗
PubMed1998

Electrostatic and structural properties of complexes involving plasmid DNA and cationic lipids commonly used for gene delivery.

UNLABELLED: The present study is aimed to characterize the interactions between plasmid DNA and cationic, large unilamellar vesicles, 110+/-20nm in size, composed of lipids commonly used for transfections including DOTAP/DOPE (mole ratio 1/1), DOTAP/DOPC (mole ratio 1/1), 100% DOTAP, or DC-CHOL/DOPE (mole ratio 1/1). [ ABBREVIATIONS: DOTAP, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; DC-CHOL, 3 beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol]. A novel approach of combining Gouy-Chapman calculations and fluorescence measurements of the pH at the surface of lipid assemblies by the fluorophore 4-heptadecyl-7-hydroxycoumarin showed that electrostatic parameters played a key role in the instantaneous formation of the DNA-lipid complexes upon addition of different amounts of plasmid DNA to cationic liposomes in 20 mM Hepes buffer (pH 7.4). Addition of large amounts of plasmid DNA leads to neutralization of 60% of the protonated DC-CHOL in DC-CHOL/DOPE (1/1) assemblies and 80% of the DOTAP in lipid assemblies. The characterization of these electrostatic parameters of the complexes suggests better and closer surrounding of plasmid DNA by lipids when DOPE is present. Time-dependent static light-scattering measurements monitored the formation of complexes and also showed that these complexes were highly unstable with respect to size at DNA/cationic lipid molar ratios between 0.2 and 0.8.

Cations↗