Purification of DNA by anion-exchange chromatography.

Column chromatography has evolved to provide a rapid and effective alternative to more laborious methods for preparing high-quality DNA, such as CsCl-gradient centrifugation. This unit describes the use of a column made of a unique anion-exchange resin that selectively binds nucleic acids, allowing rapid separation of DNA from contaminating RNA, proteins, carbohydrates, and metabolites. The procedure employs columns supplied by QIAGEN; other preparation methods are available from other suppliers. A crude nucleic acid sample (usually a cleared cell lysate) is applied to the QIAGEN tip under conditions that favor binding. Contaminants in the sample are washed from the column with a moderate-salt buffer, and DNA is eluted using a high-salt buffer.

Purchasing in a supply chain environment.

Why the interest in purchasing? In the typical company, material costs are 40% to 75% of the cost of goods sold, labor is 5% to 15%, and the balance is burden. The typical company has $4-$5 in purchased cost to $1 in labor. Most companies are implementing material requirements planning (MRP II) and enterprise resource planning systems to control the $1 in labor and have little expectation in the area of purchasing savings. Yet a dollar saved in purchasing goes directly to the bottom line. I ran a survey of 100 Class A users of MRP II; in all 100 of the companies, the biggest payback was in the area of purchasing.

Production of Plasmid DNA in Industrial Quantities According to cGMP Guidelines.

Within the last five years, the exponential growth of research activities on the development of genetic vaccination and gene therapy has made it necessary to develop an easy, cost-effective, industrial scale process for production of plasmid DNA (see Note 1). One main issue is that the process should conform to cGMP guidelines and be acceptable to the FDA or other national regulatory agencies. The cGMP environment should be implemented independently of the intended use of the DNA product.

Structure of the anchor-domain of myristoylated and non-myristoylated HIV-1 Nef protein.

Negative factor (Nef) is a regulatory myristoylated protein of human immunodeficiency virus (HIV) that has a two-domain structure consisting of an anchor domain and a core domain separated by a specific cleavage site of the HIV proteases. For structural analysis, the HIV-1 Nef anchor domain (residues 2-57) was synthesized with a myristoylated and non-myristoylated N terminus. The structures of the two peptides were studied by1H NMR spectroscopy and a structural model was obtained by restrained molecular dynamic simulations. The non-myristoylated peptide does not have a unique, compactly folded structure but occurs in a relatively extended conformation. The only rather well-defined canonical secondary structure element is a short two-turn alpha-helix (H2) between Arg35 and Gly41. A tendency for another helical secondary structure element (H1) can be observed for the arginine-rich region (Arg17 to Arg22). Myristoylation of the N-terminal glycine residue leads to stabilization of both helices, H1 and H2. The first helix in the arginine-rich region is stabilized by the myristoylation and now contains residues Pro14 to Arg22. The second helix appears to be better defined and to contain more residues (Ala33 to Gly41) than in the absence of myristoylation. In addition, the hydrophobic N-terminal myristic acid residue interacts closely with the side-chain of Trp5 and thereby forms a loop with Gly2, Gly3 and Lys4 in the kink region. This interaction could possibly be disturbed by phosphorylation of a nearby serine residue, and modifiy the characteristic membrane interactions of the HIV-1 Nef anchor domain.

Perceived effects and recovery in Oklahoma City firefighters.

This survey of 325 Oklahoma City Firefighters examined their perceptions of the effect of the bombing, their recovery and their sources of support. Other variables that were considered in this analysis included age, usefulness of the Critical Incidence Stress Management (CISM) procedures, and attitude, an aggregate variable that accounted for job satisfaction. Of particular importance in this analysis was the finding that support from "faith" was a primary predictor of positive outcome and positive attitude over the one-year period. However, the effect of the variable differed for older and younger firefighters. That is, there was a greater proportion of younger firefighters among those reporting greater support from faith. These data suggest that, at least in this geographic area, chaplains, and other spiritual leaders may play a particularly important role in the aftermath of such a disaster.

Nucleic acid immunization protects dogs against challenge with virulent canine parvovirus.

Nucleic acid vaccines (NAVs) use expression vectors encoding one or more antigen genes to transfect host cells inducing both humoral and cellular immunity against the expressed antigen. NAV offers major advantages over conventional vaccines for the protection of humans and animals. This study shows that a plasmid DNA (pGT36VP1) encoding the full length VP1 region of canine parvovirus (CPV) induces immunity that protects dogs against challenge with virulent virus. Five dogs without anti-CPV antibodies were injected at 9 months of age with increasing doses of pGT36VP1 or saline. NAV vaccinated dogs showed an increase of serum IgG titer starting 1 week post-injection which peaked at week 2 and remained detectable for at least 14 weeks. A second dose of NAV resulted in an anamnestic response within 1 week. IgG titers peaked at week 3 and 4 after the second injection. All pGT36VP1 vaccinated dogs were protected against infection after virulent CPV challenge regardless of dose and the unvaccinated control dog was fully susceptible. This study demonstrated for the first time that a NAV can protect dogs against an infectious disease.

CpG DNA is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen.

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODN) cause B cell proliferation and Ig secretion, monocyte cytokine secretion, and activation of NK cell lytic activity and IFN-gamma secretion in vivo and in vitro. The potent immune activation by CpG ODN suggests possible utility for enhancing immune responses to vaccines. Mice immunized with recombinant hepatitis B virus surface Ag and a CpG ODN as an immune enhancer have titers of Abs against HBsAg (anti-HBs) that are five times higher than those of mice immunized with HBsAg and the standard adjuvant, aluminum hydroxide (alum). Ab titers in mice immunized with HBsAg and both CpG ODN plus alum were 35 times higher than the titers in mice immunized with alum alone, indicating a strong synergistic interaction between the CpG ODN and alum. ODN without CpG motifs had little or no immune-enhancing activity at the doses used herein. Alum induces a Th2 humoral response (mostly IgG1) and no CTL. In contrast, CpG ODN gives a strong Thl response with predominantly IgG2a Abs and CTL, even when mixed with alum. In vitro studies to determine possible mechanisms of CpG immune-enhancing effects show that CpG ODN induce expression of costimulatory molecules on Ag-presenting cells and drive B cell isotype switching in the appropriate cytokine milieu. These studies demonstrate that CpG ODN are promising new immune enhancers for vaccination applications.

The role of CpG dinucleotides in DNA vaccines.

DNA vaccines can induce potent humoral and cellular immune responses without any additional adjuvant. Recent studies indicate that unmethylated CpG dinucleotides within DNA vaccines are immune stimulatory and exert an essential endogenous adjuvant activity. These CpG motifs can be added deliberately to DNA or conventional protein vaccines to enhance the Th1 immune response.

Specific cleavage sites of Nef proteins from human immunodeficiency virus types 1 and 2 for the viral proteases.

Human immunodeficiency virus type 2 (HIV-2) Nef is proteolytically cleaved by the HIV-2-encoded protease. The proteolysis is not influenced by the absence or presence of the N-terminal myristoylation. The main cleavage site is located between residues 39 and 40, suggesting a protease recognition sequence, GGEY-SQFQ. As observed previously for Nef protein from HIV-1, a large, stable core domain with an apparent molecular mass of 30 kDa is produced by the proteolytic activity. Cleavage of Nef from HIV-1 in two domains by its own protease or the protease from HIV-2 is also independent of Nef myristoylation. However, processing of HIV-1 Nef by the HIV-2 protease is less selective than that by the HIV-1 protease: the obtained core fragment is heterogeneous at its N terminus and has an additional cleavage site between amino acids 99 and 100. Preliminary experiments suggest that the full-length Nef of HIV-2 and the core domain are part of the HIV-2 particles, analogous to the situation reported recently for HIV-1.

Comparison of plasmid DNA preparation methods for direct gene transfer and genetic immunization.

Plasmid DNA is widely used for direct gene transfer in animals to study gene therapy, gene regulation, drug delivery and genetic immunization. Here we compare cesium chloride and anion-exchange purified plasmid DNA for direct gene transfer in mouse muscle and show no differences in efficiency of transfection with reporter genes or in humoral response to DNA-based immunization.

Short technical reports. Effects of lipopolysaccharide on transfection efficiency in eukaryotic cells.

The suitability of different purification methods for preparation of plasmid DNA for transfection into eukaryotic cells was systematically investigated. The reporter plasmid, pRSVcat, was prepared using several methods, and residual impurities in the preparations were quantitated. Transfection with these preparations was performed with several cell lines (HeLa, Huh7, COS7 and LMH) and two transfection methods: liposome-mediated and calcium phosphate transfection. Transfection efficiencies were determined by measuring chloramphenicol acetyltransferase expression. Higher transfection efficiencies were obtained with plasmid preparations of higher purity (those prepared by anion-exchange chromatography or two rounds of CsCl-gradient centrifugation) than with preparations of lower purity (those prepared using a silica-based DNA adsorption method or a single round of CsCl centrifugation). The results also demonstrated specifically that increasing concentrations of lipopolysaccharides in plasmid preparations directly correlate with decreasing transfection efficiencies.

Liposome mediated in vivo gene transfer into different tissues of the gastrointestinal tract.

The possibility to transfer and express genetic material in mammalian cells represents a new approach to the treatment of genetic and acquired disorders. So far, most studies use in vitro techniques to introduce foreign DNA into cultured cells, followed by reintroduction of these genetically altered cells into living organisms. In the present study we demonstrate that the LacZ marker gene can be selectively delivered, by in vivo techniques, to various locations of the gastrointestinal tract. Genetic material was targeted to the stomach, the colon, the liver and the pancreas using cationic liposomes. For transfer into the stomach and colon an intraluminal application, in the liver a portal access and in the pancreas an intraductal infusion was chosen. 48 hours after administration, the LacZ gene product beta-galactosidase could be localized in these tissues by cytochemistry. These experiments suggest a new approach to study gastrointestinal physiology and may offer novel aspects for the treatment of gastrointestinal diseases.

Gene therapy for cystic fibrosis in humans by liposome-mediated DNA transfer: the production of resources and the regulatory process.

The number of clinical trials using gene transfer technology, either active or under discussion, is increasing rapidly. However, little information is available describing the regulatory procedures or safety specifications that must be considered before initiation of such trials in Europe. We describe the procedure used by our group to produce resources for the first stage of a phase I trial of liposome-mediated gene therapy for cystic fibrosis. The current lack of written and co-ordinated guidance from the numerous interested regulatory agencies within the UK and Europe makes determination of the appropriate safety specifications and procedures for these novel trials difficult, as does the fact that some new agencies (such as the Genetic Therapy Advisory Committee in the UK) and some which are unfamiliar with clinical trials (such as the Department of the Environment) are involved as well as the Medicines Control Agency. In addition, we estimate that the realistic cost of these trials, which in many cases will have to be covered from research budgets provided by government agencies or medical charities, could lead to delays in the clinical application of this important new therapeutic strategy.

A rapid optimized protocol for downward alkaline Southern blotting of DNA.

A simple and efficient 2.5-h Southern blotting procedure is described that uses 0.4 M NaOH to transfer DNA in a downward direction. The resulting blots give signals that are both sharper and 30% stronger than those obtained by conventional upward-transferred blots.

Music and pattern change in chronic pain.

The purpose of this study was to investigate the use of music as a unitary-transformative means of altering the perception of chronic pain among women with rheumatoid arthritis within the context of Newman's model of health as expanding consciousness. In this repeated measures investigation, 30 women diagnosed with rheumatoid arthritis for a minimum of 6 months, responded to the McGill Pain Questionnaire prior to listening to music of their choice, during music, and 1 to 2 hours after completing the intervention. Data were analyzed according to the Number of Words Chosen (NWC) and the Pain Rating Index-Rank [PRI(R)] of the McGill Pain Questionnaire. The results of this study support the use of music as a unitary-transformative intervention.

Non-homologous recombination between adenovirus and AcNPV DNA fragments in cell-free extracts from insect Spodoptera frugiperda nuclei.

In previous work, we have developed a cell-free system from nuclear extracts of hamster cells to study the mechanism of integrative recombination between adenovirus type 12 (Ad12) DNA and hamster cell DNA (Jessberger et al., 1989; Tatzelt et al., 1992). We have also demonstrated that in insect cells the left terminal fragment of Ad2 DNA can insert by non-homologous recombination into the 32.6 to 34.5 map unit (EcoRI-O) fragment and into other segments of Autographa californica nuclear polyhedrosis virus (AcNPV) DNA (Xiong et al., 1991). We have now imitated this recombination event in vitro by incubating the E1 fragment of Ad2 DNA and the EcoRI-O fragment of AcNPV DNA, both in the plasmid-cloned circular forms, with partly purified nuclear extracts from Spodoptera frugiperda insect cells. Proteins from these extracts have been fractionated by gel filtration. After the reextraction of DNA from the incubation mixture, recombinants generated in this cell-free system have been identified directly with the polymerase chain reaction (PCR) by using Taq polymerase and appropriate primers which are unique to either of the two reaction partners. The recombinants identified are all different. The results of control experiments argue against the possibility that unspecific reaction products might have been generated during PCR. Nucleotide sequence determinations in some of the recombinants localize the sites of genetic exchange between the two partners and assess the non-homologous nature of the reaction. The recombinants are characterized by the presence of short patch homologies at or close to the sites of linkage between the reaction partners, as described earlier in the hamster cell system (Tatzelt et al., 1992). The occurrence of recombinants in the cell-free system can also be demonstrated by a biological test in which potential recombinants are isolated by transfection into recA- strains of Escherichia coli.

Heterologous recombination between Autographa californica nuclear polyhedrosis virus DNA and foreign DNA in non-polyhedrin segments of the viral genome.

We used the expression vector system of Autographa californica nuclear polyhedrosis virus (AcNPV) and Spodoptera frugiperda insect cells to study mechanisms of recombination in insect cells. We concentrated on the isolation and analysis of heterologous recombinants. The E1 region of human adenovirus type 2 (Ad2) was inserted into regions of the AcNPV genome which lacked apparent homologies to the polyhedrin region. Out of a total of 122 recombinant AcNPV plaques, which hybridized to Ad2 DNA in plaque annealing experiments, 13 recombinants proved heterologous, and 5 of these recombinants could be grown to titers that facilitated virus replication and further investigations of the recombinant DNA. Restriction and Southern blot analyses for all of the recombinants and nucleotide sequence determinations for one of them permitted the mapping of the sites of foreign DNA integration into the AcNPV genome for the heterologous recombinants. These sites were located in the EcoRI-C (map units 42.5-52.4), the EcoRI-L (map units 69.5-72.5), the EcoRI-O (map units 32.6-34.5), and the EcoRI-Q (map units 88.2-89.7) segments of the plaque isolate E AcNPV genome. Two of the heterologous recombinants carried the insert in the EcoRI-L fragment. The nucleotide sequence determinations across the sites of junction between the AcNPV DNA and the foreign (Ad2) DNA in one of the heterologous recombinants, AcNPV-Ad2E1-D, revealed no sequence similarities at or close to the sites of junctions. A short sequence of six nucleotides was deleted from the original EcoRI-O sequence of AcNPV at the site of insertion. The inserted Ad2E1 DNA fragment comprised nucleotides 183-2763; thus nucleotides at the termini had been deleted. In the usual polyhedrin gene-located recombinants, the foreign Ad2 DNA segment was fused to the polyhedrin promoter and recombined presumably via polyhedrin sequence segments in the vector into the polyhedrin gene of AcNPV. In one of the control recombinants, AcNPV-Ad2E1-192, the Ad2E1 DNA segment between nucleotides 1 and 3117 (out of 3322 original nucleotides) was inserted in an inverted orientation between nucleotides -115 and +735 of the polyhedrin gene of AcNPV. This particular polyhedrin sequence was deleted in the process. It was uncertain how this recombinant had been generated. The infectivities of the polyhedrin-located recombinant AcNPV-Ad2E1-192 and of the five heterologous recombinants were compared by single-cycle growth curves to the infectivity of non-recombinant AcNPV.(ABSTRACT TRUNCATED AT 400 WORDS)