Detection and quantitation of Staphylococcus aureus penicillinase plasmid deoxyribonucleic acid by reassociation kinetics.

The quantity of penicillinase plasmid deoxyribonucleic acid (DNA) in various strains of Staphylococcus aureus has been determined by DNA-DNA reassociation kinetics. Specifically, 32P- or 125I-labeled denatured probes of purified plasmid DNA were reassociated in the presence of denatured DNAs isolated from the bacterial strains in question. The number of plasmid copies per cell was calculated from the effect of the latter nucleic acid samples on the reassociation rate of the radiolabeled probe. Among the S. aureus strains examined were monoplasmid, diplasmid and replication-defective representatives, and the effect of temperature on wild-type plasmid content was also investigated.     

Pharmacological activity of echinochrome a alone and in the biologically active additive Timarin

Pharmacological activity of echinochrome A (EchA) alone and in the biologically active additives (BAA) Timarin, administered per os has been investigated in volunteers. Blood hematological, immunological, and biochemical parameters were investigated before and after administration of the substances used. EchA decreased serum glutatione (GSH) and increased catalase activity 1 h after treatment. Later (3 h after administration) catalase activity normalized, while GSH exceeded the initial level. Changes in blood lipids suggest decreased risk of atherogenesis. Changes found in blood sex hormone levels indicate that Timarin may influence sex gland functioning. Changes of hematological and immunological parameters have been interpreted as the result of a mild stressor effect of both EchA alone and in the BAA Timarin increasing adaptation reactivity of the body.     

Ascorbate/iron mediation of hydroxyl free radical damage to PBR322 plasmid DNA

Plasmid PBR322 DNA has been exposed to hydroxyl free radicals generated from an ascorbate/Fe system. Hydroxyl free radical scavengers as well as the iron chelator desferroxamine and catalase inhibit the DNA nicking which occurs, but superoxide dismutase had no effect. The DNA nicking was temperature dependent, occuring more rapidly at higher temperatures. The rate of DNA nicking was accelerated by the addition of hydrogen peroxide. There was an early lag phase in DNA nicking, even though the rate of hydroxyl free radical generation, as assessed by salicylate hydroxylation, showed no lag phase. It is considered that the early hydroxyl free radical damage to DNA may be biologically very important in mutagenic and carcinogenic processes.     

Biomedical application of plasmid DNA in gene therapy: A new challenge for chromatography

Abstract

Gene therapy and DNA vaccination are clinical fields gradually emerging in the last few decades, in particular after the discovery of some gene-related diseases. The increased relevance of biomedical applications of plasmid DNA (pDNA) to induce therapeutic effects has had a great impact on biopharmaceutical research and industry. Although there are several steps involved in the pDNA manufacturing process, the several unit operations must be designed and integrated into a global process. After the plasmid has been designed according to the requirements for clinical administeration to humans, it is biosynthesised mainly by an E. coli host. The overriding priority of the production process is to improve plasmid quantity - the production conditions need to be optimised to guarantee pDNA stability and biological activity.

The complexity and diversity of biomolecules present on the pDNA-containing extracts represent the main concern and limitation to achieve pure and biologically active pDNA. There has been a recent intenstification of the improvement of existing purification procedures or the establishment of novel schemes for plasmid purification.

In spite of the efficacy to purify sc pDNA, these matrices present relatively low binding capacities. Hence, the application of large pore matrices in order to further increase capacity and open the way to process scale applications could be a great advantage for affinity chromatography.     

Rapid isolation of high quality, multimeric plasmid DNA using zwitterionic detergent

Purification of plasmid DNA from bacteria is an essential tool in recombinant DNA technology and has become an essential task in laboratories to industries. Moreover, the recent progress of "Gene therapy" and "Genetic vaccination" also demands production of pharmaceutical grade plasmid DNA in 'kilogram' level. Despite existence of a number of purification protocols, all most all have been originated from a pioneering work [Birnboim, H.C., Doly, J., 1979. A rapid extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513-1523] and so suffer from one or more drawbacks, such as purification time, purity or quantity of isolated plasmid DNA. Here, we have reported an innovative approach for isolation of highly pure and functional plasmid DNA in significant amount, based on generation of "soft protein aggregate" with the help of zwitterionic detergents and alkali. Solibilized proteins and RNA could be removed by a simple and mild washing with Tris buffer of low ionic strength and multimeric plasmid DNA could be eluted in a single step from the protein aggregate. Additionally, isolated plasmid DNA could easily be digested by restriction enzymes and had high functionality in protein expression. Thus, considering both its remarkable simplicity and efficiency in producing sufficiently pure plasmid DNA, the new strategy would emerge a useful tool in modern recombinant technology and therapeutic applications.     

Expression of kringle 5 domain of human plasminogen in Pichia pastoris

The kringle 5 domain of plasminogen exhibits potent inhibitory effect on endothelial cell proliferation. It can also cause cell cycle arrest and apoptosis of endothelia cell specifically, and shows promise in antiangiogenic therapy. It has been prepared via both proteolysis of native plasminogen and recombinant DNA methodologies. When expressed in E. coli, recombinant, kringle 5 deposited mainly as inactive, insoluble inclusion bodies and the refolding yield was also low. In the present study, human kringle 5 encoding gene was cloned into secretory plasmid pPIC9K and then integrated into Pichia pastoris genome for expression. On methanol induction, biologically active recombinant kringle 5 was expressed and secreted into the culture medium by the integrated Pichia pastoris with the expression level around 30mg/L of yeast culture. After a simple and economical three-step purification protocol, namely precipitation, DEAE ion exchange chromatography, and gel filtration, the recombinant kringle 5 was purified to homogeneity, with the yield of 7.5 mg/liter yeast culture.     

A novel method of plasmid isolation using laundry detergent

Since the discovery of plasmid, various methods have been developed to isolate plasmid DNA. All the methods have one common and important target of isolating plasmid DNA of high quality and quantity in less time. These methods are not completely safe because of use of toxic chemicals compounds. The developed protocol for plasmid extraction is based on the alkaline lysis method of plasmid preparation (extraction atpH 8.0) with slight modifications. Cell lysis reagent sodium dodecyl sulfate is replaced by lipase enzyme present in laundry detergent. A good plasmid preparation can be made, which is well suited for subsequent molecular biology applications. By taking safety measures on count, contaminants like, RNA and protein can be completely avoided with maximized plasmid yield. The resultant plasmid quality and quantity can be well comparable to other prevalent methods.     

Protective effects of catalase overexpression on UVB-induced apoptosis in normal human keratinocytes

UV-induced apoptosis in keratinocytes is a highly complex process in which various molecular pathways are involved. These include the extrinsic pathway via triggering of death receptors and the intrinsic pathway via DNA damage and reactive oxygen species (ROS) formation. In this study we investigated the effect of catalase and CuZn-superoxide dismutase (SOD) overexpression on apoptosis induced by UVB exposure at room temperature or 4 degrees C on normal human keratinocytes. Irradiation at low temperature reduced UV-induced apoptosis by 40% in normal keratinocytes independently of any change in p53 and with a decrease in caspase-8 activation. Catalase overexpression decreased apoptosis by 40% with a reduction of caspase-9 activation accompanied by a decrease in p53. Keeping cells at low temperature and catalase overexpression had additive effects. CuZn-SOD overexpression had no significant effect on UVB-induced apoptosis. UVB induced an increase in ROS levels at two distinct stages: immediately following irradiation and around 3 h after irradiation. Catalase overexpression inhibited only the late increase in ROS levels. We conclude that catalase overexpression has a protective role against UVB irradiation by preventing DNA damage mediated by the late ROS increase.     

High level expression of kringle 5 fragment of plasminogen in <Emphasis Type="Italic">Pichia</Emphasis><Emphasis Type="Italic">pastoris</Emphasis>

Angiogensis can be blocked by inhibitors such as endostatin and angiostatin. The kringle 5 fragment of plasminogen also has a potent inhibitory effect on endothelial cell proliferation and leads to the inhibition of angiogenesis. It has promise in anti-angiogenic therapy due to its small size and potent inhibitory effect. Preparation of kringle 5 has been achieved through the proteolysis of native plasminogen and recombinant DNA technology. Bacterially expressed recombinant kringle 5 is mainly insoluble and expressed at low level. The refolding yield is also low. To produce recombinant human kringle 5 in a large quantity, we have genetically modified a strain of Pichia pastoris. On methanol induction, this strain expressed and secreted biologically active, recombinant kringle 5. The expression level of the engineered strain in culture reached more than 300mgl^-1. Purification was easily achieved by precipitation, hydrophobic and DEAE ion exchange chromatography. The recovery of recombinant kringle 5 was about 50% after purification. Yeast-expressed kringle 5 has a higher activity in anti-endothelial proliferation than bacterially expressed kringle 5.Revisions requested 9 November 2004; Revisions received 2 December 2004     

Sensitized NADH formation of single-stranded breaks in plasmid DNA upon the action of near UV-radiation

It has been shown that NADH photosensitize in vitro single-strand breaks formation in double-strand plasmid DNA pBR 322 upon near-UV (320-400 nm) irradiation. The number of single-strand breaks depends both on UV light dose and sensitizer concentration. Addition of catalase and sodium benzoate strongly decreases the single-strand breaks formation. The results show an important role of hydrogen peroxide (H2O2) and hydroxyl radical (.OH) in inducing single-strand breaks in plasmid DNA irradiated by near-UV radiation in the presence of NADH.     

Transduction of plasmid determinants in Staphylococcus aureus and Escherichia coli.

Buoyant density analysis of transducing lysates derived from Staphylococcus aureus and Escherichia coli indicated that phage particles bearing plasmid determinants contain a quantity of DNA equivalent to that found in the lytic particles. Transducing particles that bear plasmid determinants smaller than viral DNA must therefore contain a quantity of DNA in excess of a single plasmid genome. In the E. coli P1vir system, a dependence upon host-mediated recombination for the transduction of small plasmids, but not for large R factors or chromosomal genes, was observed. However, no evidence for the involvement of such functions in the transduction of S. aureus plasmids was obtained. Although the origin of the additional DNA in plasmid transducing particles has not been identified, circumstantial evidence has been presented in the staphylococcal system indicating that transducing particles carrying a small tetracycline plasmid are not formed by the wrapping of multiple copies of this plasmid DNA.     

Regulatory role of low-intensity laser radiation on the status of the antioxidant system

On the base of modern literary data it is shown that low level laser red radiation can render the manifested effect on the activity of main antioxidant enzymes. It is determined that the main enzymes of antioxidant system--superoxide dismutase, catalase, ceruloplasmine--absorb in the red part of spectrum and are capable to be reactive under some regimes of red low level laser irradiation. It is one of the main possible mechanisms of biological efficiency of low level red laser radiation of the red part of spectrum.     

Replication of the Bacteriocinogenic Plasmid Clo DF13 in Thermosensitive Escherichia coli Mutants Defective in Initiation or Elongation of Deoxyribonucleic Acid Replication

The replication of the bacteriocinogenic plasmid Clo DF13 has been studied in the seven temperature-sensitive Escherichia coli mutants defective in deoxyribonucleic acid (DNA) replication (dnaA-dnaG). Experiments with dna initiation mutants revealed that the replication of the Clo DF13 plasmid depends to a great extent on the host-determined dnaC (dnaD) gene product, but depends slightly on the dnaA gene product. The synthesis of Clo DF13 plasmid DNA also requires the dnaF and dnaG gene products, which are involved in the elongation of chromosomal DNA replication. In contrast, the Clo DF13 plasmid is able to replicate in the dnaB and dnaE elongation mutants at the restrictive temperature. When de novo protein synthesis is inhibited by chloramphenicol in wild-type cells, the Clo DF13 plasmid continues to replicate for at least 12 h, long after chromosomal DNA synthesis has ceased, resulting in an accumulation of Clo DF13 DNA molecules of about 500 copies per cell. After 3 h of chloramphenicol treatment, the Clo DF13 plasmid replicates at a rate approximately five times the rate in the absence of chloramphenicol. Inhibition of protein synthesis by chloramphenicol does not influence the level of Clo DF13 DNA synthesis at the restrictive temperature in the dna mutants, except for the dnaA mutant. Chloramphenicol abolishes the inhibition of Clo DF13 DNA synthesis in the dnaA mutant at the nonpermissive temperature. Under these conditions, Clo DF13 DNA synthesis was slightly stimulated in the first 30 min after the temperature shift, and continued for more than 3 h at an almost uninhibited level.     

Effects of salt and temperature on plasmid topology in the halophilic archaeon Haloferax volcanii.

We report here the effect of environmental parameters, salinity, temperature, and an intercalating drug on plasmid topology in the halophilic archaeon Haloferax volcanii. We first studied the topological state of the plasmid pHV11 in media of different salt compositions and concentrations. The superhelical density of plasmid PHV11 varies in a way that depends on the kind of salt and on the concentrations of individual salts. With respect to growth temperature, the plasmid linking number increased at higher temperature in a linear way, contrary to what has been reported for Escherichia coli, in which the plasmid linking number decreased at higher temperature. These results suggest that some of the mechanisms that control DNA supercoiling in halophilic Archaea may be different from those described for E. coli. However, homeostatic control of DNA supercoiling seems to occur in haloarchaea, as in Bacteria, since we found that relaxation of DNA by chloroquine triggers an increase in negative supercoiling.     

Release of transforming plasmid and chromosomal DNA from two cultured soil bacteria

The release of chromosomal and plasmid DNA from Acinetobacter calcoaceticus and Bacillus subtilis cultivated in minimal medium and broth over a period of 50 h was monitored and related to growth phase, autolysis, DNase production and natural competence. The released DNAs were biologically active in natural transformation. In addition, the circular integrity of a released B. subtilis shuttle vector (pHV14) was demonstrated by artificial transformation of Escherichia coli. In cultures of both strains high molecular weight DNA accumulated, particularly during the stationary and death phase (up to 30 g ml^-1). Generally, despite the presence in culture fluids of DNase activity (and of an intracellular enzyme, catalase, indicating some cell lysis) there was high transforming activity of chromsomal and plasmid DNA even 40 h after the cultures reached the stationary phase. In cultures of B. subtilis in minimal medium a presumably active release of intact plasmids and chromsomal DNA occurred during the competence phase. The release of biologically functional DNA during essentially all growth phases of a gram-positive and a gram-negative member of soil bacteria might facilitate horizontal gene transfer by transformation in natural habitats.     

Induced mutagenesis of plasmid and chromosomal genes inserted into plasmid DNA. I. The mutagenic action of radiation

This paper describes the results of treating plasmid DNA in vitro with mutagens, to obtain mutations both in plasmid genes and chromosomal genes comprised within the plasmid, thus avoiding disorganization characteristic of in vivo mutagenesis. The model system is represented by DNA of RSF2124 responsible for colicine E1 synthesis and resistance to ampicillin. Col- mutants were looked for after exposure to UV- and gamma-irradiation. The lethal effect was estimated as inactivation of the ampicillin resistance marker. After reisolation from mutant transformant of the plasmid DNA, the novel character and resistance to ampicillin proved to retain in the course of subsequent transformations and passages of transformed colonies, suggesting the mutational nature of the changes. Exposure of RSF2124 to short-wave UV-irradiation (lambda = 254 nm) produced a pronounced mutagenic effect: the relative quantity of Col- mutants under optimal conditions of mutagenesis increased about 10 times. In the case of W-reactivation (additional UV-irradiation of C600 wild type cells) of lethal lesions, a 95% reliable increase in mutagenic effect was observed. Significant enhancement of mutagenesis (about 4-fold) was detected when only recipient cells were exposed to low doses of UV (the so-called indirect UV mutagenesis). Thus, with regard to W- and indirect UV mutagenesis, the plasmid DNA behaves like DNA of temperate phages which suggests their evolutionary relationship. Treatment of plasmid DNA with acridine orange prior to UV, only protected from lethal lesions. Gamma-irradiation (60Co) at the dose producing 100-fold inactivation, increased the yield of Col- mutants by one order of magnitude. The presence of RSF2124 plasmid in a cell does not affect its UV sensitivity.     

The oxidative damage of plasmid DNA by ascorbic acid derivatives in vitro: the first research on the relationship between the structure of ascorbic acid and the oxidative damage of plasmid DNA

To study the structure-function relationship of the oxidative-damage effect of ascorbic acid, we have focused on the interaction between plasmid DNA pUC19 and a series of ascorbic acid derivatives modified on different OH groups in the presence of transition metal ions. Some ascorbic acid derivatives can selectively cleave plasmid DNA from Form I to Form II in the presence of low concentration of Cu2+ just like ascorbic acid itself, while other derivatives oxidatively damage plasmid DNA slightly. We found that those derivatives with unattached 2-OH and 3-OH groups retain the ability to cleave the plasmid DNA. The derivatives that have been methylated on 2-OH or 3-OH can only cleave plasmid DNA softly, and those derivatives that have been protected on both 2-OH and 3-OH can hardly exert an oxidative damage on plasmid DNA under the same condition. Form these results, we can draw the conclusion that 2-OH and 3-OH groups of the ascorbic acid molecule contribute most to this biological activity.     

The chromosomal integration site for the Streptomyces plasmid SLP1 is a functional tRNATyr gene essential for cell viability

The genetic element SLP1 exists in nature as a single DNA segment integrated into the genome of Streptomyces coelicolor. Upon mating with Streptomyces lividans, a closely related species, SLP1 undergoes precise excision from its chromosomal site and is transferred into the recipient where it integrates chromosomally. Previous work has shown that integration and excision involve site-specific recombination between a chromosomal site, attB, and a virtually identical sequence, attP, on SLP1. We demonstrate here by means of gene replacement that a tRNA(Tyr) sequence that overlaps part of the attB site of S. lividans is both biologically functional and essential for cell viability. The requirement for this tRNA gene has been used to stabilize the inheritance of a segrationally unstable plasmid in cells lacking a chromosomal attB site. The evolution of an essential DNA locus as an attachment site for a chromosomally integrating genetic element represents a novel mechanism of biological adaptation.     

Characterization of a topologically aberrant plasmid population from pilot-scale production of clinical-grade DNA

As part of a program to develop DNA vaccines for pharmaceutical applications, we recently established a manufacturing process for the production of clinical grade plasmid DNA. In an evaluation of two cell separation methods, the cell culture experienced a temperature spike in a new tangential flow filtration rig, resulting in an aberrant plasmid HPLC peak. Analysis by agarose gel electrophoresis and HPLC demonstrated that the aberrant plasmid material's overall primary structure, methylation pattern and topological integrity was indistinguishable from that of reference material. Transmission electron microscopy and high-resolution agarose gel electrophoresis revealed that the unknown plasmid form exhibited a very low level of supercoiling, whereas the normal supercoiled fraction contained highly twisted DNA. We hypothesized that an enzymatic process, induced by stress during the temperature spike, caused the distinct plasmid topology. This idea was supported by a lab-scale fermentation experiment, where plasmid topology was shown to be similarly altered by conditions designed to induce metabolic stress.