Treated Plasmid DNA Research Articles

The integrity of chemically treated plasmid DNA as a chemical-based choice for prion clearance.

In regenerative medical products for clinical applications, a major concern is the risk of ruminant-derived materials developing transmissible spongiform encephalopathy (TSE) in the manufacturing process. Because of the risk of TSE causing prion disease, the raw materials derived from ruminants should be compliant with the “Standard for Biological Raw Materials” to ensure the quality and safety of pharmaceutical products. We therefore tested whether plasmid DNA could withstand four chemical reagents (Gdn-HCl, Gdn-SCN, TCA, or SDS), having referred to the report by Tateishi et al. [1], which describes how Creutzfeldt–Jakob disease pathogens can be inactivated by chemical reagents capable of producing a 7-log reduction in prion inactivation. We observed that plasmid DNA was mixed with chemical reagents and that the functionality of plasmid DNA was equivalent for both chemical and non-chemical treatment. The potency of plasmid DNA was monitored by the existence of DNA fragments and the function by which GFP proteins were produced by HEK293-cell transfected plasmid DNA. The existence of DNA fragments was detected in plasmid DNA treated by chemical reagents, except when undergoing TCA treatment. Additionally, when HEK293 cells were transfected with the plasmid DNA after chemical treatment, GFP protein was produced. These results indicate that plasmid DNA can withstand the chemical treatments for blocking prion transmission.

Screening of Phytochemical Compounds and Assessment of Antiurolithiatic Activity of Pisonia Alba Leaves Extracts against DNA Damage

This work investigate the Phytochemical constituents of Pisoniaalba (PA) leaves using standard protocols and to determine its DNA damage inhibition and Antiurolithiatic capability. Dried leaves of Pisoniaalba were used to screen and quantify the phyto constituents in different organic solvents extracts that are analysed by GCMS, FTIR and HPTLC. In the presence of PUC18 plasmid DNA, the DNA damage inhibition assay was performed by photolysing H2O2 with UV radiation and agarose gel electrophoresis with irradiated DNA. The invivo antiurolithic assay was performed by nucleation method. Pisoniaalbain's phytochemical analysis reveals the existence of secondary metabolite components including glycosides, resins, phenols, terpenoids, flavonoids, tannins, steroids, and alkaloids, etc.. ELPA express the maximum quantity of phenols, flavonoids, alkaloids and terpenoids which were 30.44 ±0.65 mg TAE/g extract, 28.51 ± 1.19 mg RE/g extract, 28.08 ± 0.08 mg of AE/g of extract, 29.94 ± 0.32 mg RE/g extract respectively. The GC-MS assessment findings confirmed the presence of nine ELPA phyto compounds accompanied by eleven ALPA phytoc ompounds. It was also observed that Pisoniaalba leaves prevents DNA damage in UV and H2O2 treated plasmid DNA. The invitro Antiurolithiatic research indicates that ELPA was more efficient in blocking calcium oxalate nucleation. From the obtained results it was proven that the Pisonia alba leaves has potential secondary metabolites with therapeutic role mainly against urolithiasis. Both invitro and in vivo analysis demonstrates that ELPA significantly reduces crystal formation that are causative of renal stones. These evidences suggest that Pisoniaalbacan be further investigated for the prevention and treatment of urolithiais

Quantitative analysis of oxidative DNA damage induced by high-voltage pulsed discharge with cavitation

Abstract Pulsed discharge is used for sterilization and disinfection, but the details of the molecular mechanisms remain largely unknown. Since pulsed discharge generates reactive oxygen species (ROS), we analyzed the oxidative DNA damages after pulsed discharge treatment to consider the involvement of ROS in the damaging process. We applied pulsed discharge with cavitation to plasmid DNA molecules and estimated the yields of the damages by agarose gel electrophoresis. The treated DNA contained various oxidative DNA damages, including single and double strand breaks and base lesions. The yields of the damages increased in response to the energy used for pulsed discharge. We also measured the yield of 8-hydroxyguanine (8-OH-G), one of the major oxidative base lesions, in the treated plasmid DNA by mass spectrometry quantitatively and found that the yield of the oxidative base lesion corresponded to the increment of the applied energy. In addition, we observed the involvement of mutM gene, which is responsible for repair of 8-OH-G, in the increased sensitivity of Escherichia coli to pulsed discharge. Therefore, ROS seem to mediate the sterilization ability of pulsed discharge.

Comparison of the Effectiveness of Chlorine, Ozone, and Photocatalytic Disinfection in Reducing the Risk of Antibiotic Resistance Pollution

AbstractEffectiveness of conventional chlorine and ozone disinfection on reduction of antibiotic resistance was compared with less commonly applied heterogeneous photocatalytic process. For this purpose plasmid DNA isolated from a multi-resistant Escherichia coli (E. coli) HB101 was treated in two different concentrations with the three oxidation processes. Oxidative damage on the plasmid DNA was analyzed with gel electrophoresis by comparing the extent of conformational changes in the DNA structure. The effectiveness of the applied oxidant in reducing the risk of resistance transfer was also evaluated by comparing the ability of treated plasmid DNA to transform competent cells. Chlorine did not affect plasmid DNA structure at the studied doses, while ozone and photocatalytic treatment resulted in conformational changes and the damage increased with increasing oxidant doses. Transformation experiments confirmed a similar trend. Chlorine did not affect the transformability and the cell counts of competent cells transformed with chlorine treated plasmid DNA were similar to those transformed by non-treated plasmid DNA in the control experiments.

Effects of Non-thermal Plasmas on DNA and Mammalian Cells

The application of non-thermal (cold) plasmas in medicine holds great promise. Non-thermal plasma devices have been used to directly ablate cells, induce apoptosis, facilitate cell transfection, lay down bioscafolds, and sterilize heat sensitive materials. Although the reactive species (RS) generated by the plasmas have been implicated in all of these processes, the biological mechanisms of action are poorly understood and the potential adverse effects largely unknown. In this work, we used a parallel electrode dielectric barrier discharge (DBD) and the atmospheric pressure glow discharge torch (APGD-t) to assess the possible negative effects of direct and indirect plasma treatment of mammalian cells and naked DNA. HeLa cells demonstrated an oxidative stress response when placed in direct contact with the plasma. No lipid peroxidation resulted from the application of the plasma to the cells. Both plasma sources were shown to be able to fragment naked DNA in PBS and to cause DNA double-strand breaks (DSBs). Sequencing of treated plasmid DNA introduced into electrocompetent bacterial cells showed no evidence of mutations. We conclude that the reactive nature of non-thermal plasmas can create oxidative stresses and may have unintended negative biological effects on cells.

A method to increase the bioactivity of plasmid DNA by heat treatment

A method to increase the bioactivity of plasmid DNA by heat treatment has been developed. The structure of the heat treated plasmid DNA was investigated by electrophoresis assay and atomic force microscope (AFM) observation. Electrophoresis assay showed that the heat treated DNA consisted of three components: the supercoiled DNA (component I), the open circular DNA (component II) and the heat denatured DNA component. The bioactivity of the heat treated plasmid DNA was investigated by both DNA condensation experiments and gene transfection experiment with mammal cells. DNA condensation experiments showed that the heat denatured DNA component owned higher sensitivity to spermidine and polyethylenimine (PEI) than component I and component II DNA. Gene transfection experiment with PEI indicated that the heat treated DNA had higher gene transfection efficiency than untreated DNA. Our experiment not only shows an effective approach to increase the bioactivity of plasmid DNA but also leads a way to improve the bioactivity of DNA by physically modifying their structure.

Mechanisms of chromium-induced suppression of RNA synthesis in cellular and cell-free systems: relationship to RNA polymerase arrest.

Chromium(VI) (Cr(VI)) can suppress both DNA replication and transcription as a result of chromium (Cr)-induced DNA damage. While progress has been made in the characterization of Cr-induced DNA polymerase arresting lesions, very little information is available on the inhibition of transcription by this metal. The aim of the present study was to identify the molecular mechanisms involved in the reduction of RNA synthesis by Cr. Following treatment with a moderately cytotoxic dose (approximately LC50) of Cr(VI) (150 microM for 2 h), total RNA synthesis was initially suppressed in CHO cells and recovered to control levels within 72 h post-treatment. In vitro nuclear run-on transcription assays of nuclei isolated from Cr(VI)-treated cells showed a similar amount of RNA synthesis suppression as observed in intact cells. Qualitative analysis of nascent transcripts revealed a general, concentration-dependent reduction in size suggesting that transcriptional elongation was inhibited following Cr-treatment. Transcriptional initiation in these nuclei was also reduced. To better determine whether transcriptional suppression was related to Cr-induced DNA damage we examined the transcriptional activity of T7 RNA polymerase on Cr(III)-treated plasmid DNA. Treatment of pGEM3Z-TS DNA with Cr(III) resulted in transcriptional arrest which occurred primarily at GC-rich and palindromic regions. However, in contrast to the cellular data, transcriptional initiation was unaffected in the in vitro transcription arrest assays. Taken together, these results suggest that the suppression of RNA synthesis by Cr is related to chromium-induced template DNA damage which prevents elongation leading to premature RNA polymerase arrest.

Iron chelators modulate the production of DNA strand breaks and 8-hydroxy-2′-deoxyguanosine

The interaction of chelators and reducing agents is of particular importance in understanding iron-associated pathology since catalytic iron undergoes cyclic reduction and oxidation in vivo. Therefore, we treated plasmid DNA with free or chelated Fe(III) in the presence of biological reductants, and simultaneously measured the number of single strand breaks (SSBs) and oxidative base modification (8-hydroxy-2′-deoxy-guanosine; 8-OHdG) by quantitative gel electro-phoresis and HPLC with electrochemical detection, respectively. Production of SSBs and 8-OHdG was linearly correlated suggesting that these two different lesions share a common chemical mechanism. The levels of both lesions were enhanced when Fe(III) was chelated to citrate or nitrilotriacetic acid. Reducing agents showed different potency in inducing DNA damage catalyzed by chelated iron (L-ascorbate > L-cysteine > H2O2). Chelation increased SSB formation by ∼ 8-fold and 8-OHdG production by ∼ 4-fold. The ratio of SSB/8-OHdG catalyzed by chelated iron, which is twice as high as by unchelated iron, indicates that chelation affects iron-catalyzed oxidative DNA damage in a specific way favoring strand breakage over base modification. Since iron is mostly chelated in biological systems, the production of genomic and mitochondrial DNA damage, particularly strand breaks, in diseases involving iron overload is likely to be higher than previously predicted from studies using unchelated iron.

Differential Sensitivity of Chromium-Mediated DNA Interstrand Crosslinks and DNA-Protein Crosslinks to Disruption by Alkali and EDTA

Some compounds of hexavalent chromium are well-established carcinogens. Chromium enters mammalian cells in the hexavalent form and is reduced to chromium(III). Treatment of purified DNA with chromium(III) produces DNA-DNA inter-strand crosslinks (DDC) which obstruct the progression of DNA polymerases in vitro. DDC were also detected in chromate-treated cultured normal human lung cells using the renaturing agarose gel electrophoresis (RAGE) assay and correlated with base-specific inhibition of DNA replication. Curiously, DDC have gone undetected in studies of cultured cells using the alkaline elution (AE) technique, whereas chromium-mediated DNA-protein crosslinks (DPC) were readily detected by AE. We tested the hypothesis that AE conditions [60 mM tetraethyl ammonium hydroxide (TEA), 20 mM EDTA, pH 12.6, for 16 h at room temperature] dissociate DDC but not DPC using chromium(III)-treated plasmid DNA and the RAGE assay. Dose-dependent chromium-induced DDC were unaffected by TEA (pH 11.8) alone or by more rigorous alkaline denaturation conditions (200 mM NaOH, pH 13.5, for 16 h). DDC were, however, completely disrupted by EDTA (pH 12.6) alone or the combination of TEA and EDTA (pH 12.6). In contrast, DPC remained largely intact under these conditions. Therefore, past AE-based studies which have failed to detect chromium-induced DDC do not prove the absence of this lesion. AE may not be suitable for detecting DDC induced by EDTA-chelatable agents such as metals.

Mutagenic and recombinagenic effects of diethylstilbestrol quinone

Estrogens are believed to be major contributors to many cancers of the human female genital tract, but the mechanism of their carcinogenic action is not well-understood. While a tumor-promoting role for estrogens is well-supported, whether they also act as tumor initiators has remained controversial. Here, we have sought to examine the mutagenic potential of diethylstilbestrol, a synthetic estrogen that is a powerful carcinogen in hamsters, and is suspected to be a human carcinogen. Phage M13 single-stranded DNA was treated in vitro with diethylstilbestrol quinone (DES Q; 1.25 mM) and transfected into Escherichia coli cells. DES Q treatment resulted in an apparent enhancement of mutagenesis in the LacZ(α) gene segment. DNA sequence analysis of LacZ(α) mutants obtained by transfection of DES Q-treated DNA revealed that the major effect of DES Q treatment has been a 6-fold elevation of recombination between the phage-borne LacZ(α) sequence and the LacZΔM15 sequence on the E. coli fertility plasmid F. To confirm whether DES Q treatment is recombinagenic, we used an experimental system that allows the detection of recombination between a defective E. coli chromosomal LacY gene and a normal counterpart borne on a plasmid. Transfection of DES Q (0.06–12 mM) treated plasmid DNA showed significant enhancement (2-100-fold) in recombination, but not in mutagenesis. These results raise the possibility that estrogen quinones may induce recombinagenic DNA damage.

Mutagenesis of plasmid DNA with hydroxylamine: isolation of mutants of multi-copy plasmids.

An investigation of in vitro mutagenesis of plasmid DNA with hydroxylamine is described. The treated plasmid DNA was used to transform Escherichia coli K12. Mutants of the plasmid NTP3, which codes for resistance to ampicillin and sulphonamides, were isolated and characterised. They were classified according to the reduction in level of their beta-lactamase activity. Hydroxylamine-induced mutants of NTP14 were also isolated. This plasmid codes for ampicillin resistance, synthesis of colicin E1, and the EcoRI restriction and modification enzymes. One class of mutants is lethal to the host strain at temperatures above 33 degrees C, but carrier strains grow well at 28 degrees C. There is evidence that these mutants code for a temperature-sensitive EcoRI modification activity: the lethal effect probably results from the cleavage of the host-cell DNA by the restriction enzyme at non-permissive temperatures. The possible genetic uses of the mutant plasmids for the production of hybrid plasmids in the bacterial cell are discussed.