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Biomedical subjects

E Wagner

Publications and source records attributed to E Wagner.

At least 19 recordsLinked to original sources
PubMed1995

Binding of adenylate kinase to RNA.

Adenylate kinase (ATP:AMP transphosphorylase) is a key enzyme in energy metabolism. The activity of its isoforms is subjected to multiple regulations. It is shown here that a specific fraction consisting of all adenylate kinase isoforms from tobacco leaves and tissue cultures does not bind to the anionic exchange-resin Mono Q. Sample pretreatment with ribonuclease could restore full binding to Mono Q, suggesting an association of adenylate kinase with RNA similar to the enzyme of Chenopodium rubrum (J. Chromatogr. 625: 13-19). We propose here that at least in vitro adenylate kinase can behave as an RNA-binding protein and that RNA-binding of adenylate kinase isoforms may be related to regulatory mechanisms.

Adenylate Kinase↗
PubMed1995

Evaluating community-based nutrition programs: comparing grocery store and individual-level survey measures of program impact.

BACKGROUND: This paper examines whether an "environmental indicator"--a survey of grocery store product displays--can provide a realistic alternative to individual-level telephone surveys for the evaluation of community-based nutrition programs. METHODS: Telephone and grocery store measures were used separately to evaluate three community-level dietary interventions that were part of the Kaiser Family Foundation Community Health Promotion Grants Program (CHPGP). Both surveys were conducted in the three intervention and seven control communities at three points in time: 1988, 1990, and 1992. The grocery store survey recorded the relative availability of low-fat and high-fiber products and the amount of store-provided health-education information. Self-reported dietary intake of residents was obtained in the same communities using a telephone survey. RESULTS: In the one community in which the intervention seemed to have contributed to reduced fat consumption, the grocery store and telephone surveys showed very similar relative changes for the only variable they had in common, low-fat milk consumption. In another community, both survey approaches indicated that there was no change or perhaps a slight worsening in the treatment relative to the controls. The third community produced the only contradictory results: the telephone survey suggested no change or perhaps a worsening, while the grocery store results were generally positive, though not statistically significant. CONCLUSION: These results, combined with the much lower cost of the grocery store survey, justify further pursuit of environmental indicators as an evaluation tool.

Diet↗
PubMed1995

Virus-mediated release of endosomal content in vitro: different behavior of adenovirus and rhinovirus serotype 2.

Endosomal penetration by nonenveloped viruses might be accomplished by either local breakdown of the endosomal membrane (e.g., adenovirus) or formation of a membrane-spanning pore by capsid proteins. Uncoating of the nonenveloped virus human rhinovirus serotype 2 (HRV2) has been shown to occur from late endosomes and to be entirely dependent on the acidic pH in this compartment (Prchla, E., E. Kuechler, D. Blaas, and R. Fuchs. 1994. J. Virol. 68: 3713-3723). To investigate further the mechanism of uncoating of HRV2, an in vitro assay was established to test viruses or virus-derived peptides for their capacity to release cointernalized biotin-dextran of different molecular mass (10 and 70 kD) from isolated endosomes. The suitability of the assay was demonstrated by use of a fusogenic peptide derived from influenza virus hemagglutinin (GALA-INF3). Whereas adenovirus induced a low pH-dependent release of up to 46% of the internalized biotin-dextran and did not show any significant size selectivity (as expected for endosome disruption), HRV2 mediated release of 27% of the 10 kD dextran and only traces of the 70-kD dextran. Similarly, GALA-INF3-induced release of biotin-dextran was also size dependent. The potential role of the capsid protein VP1 in HRV2 uncoating in vivo was also substantiated in our in vitro system using an amphipathic, NH2-terminal peptide of VP1. Taken together, these data favor the model of a specific pore-forming mechanism for HRV2 uncoating which is in contrast to the membrane-disrupting mechanism of adenovirus.

Adenoviridae↗
PubMed1995

Successful histocompatible myoblast transplantation in dystrophin-deficient mdx mouse despite the production of antibodies against dystrophin.

Myoblast transplantation has been considered a potential treatment for some muscular disorders. It has proven very successful, however, only in immunodeficient or immunosuppressed mice. In this study, myoblasts from C57BL10J +/+ mice were transplanted, with no immunosuppressive treatment, in the tibialis anterior of fully histocompatible but dystrophin-deficient C57BL10J mdx/mdx mice. One to 9 months after transplantation, the success of the graft was evaluated by immunohistochemistry. All the transplanted mice (n = 24) developed dystrophin-positive fibers following transplantation. Depending on myoblast cultures, transplantations, and time of analysis, the mice presented 15 to 80% of dystrophin-positive fibers in transplanted muscles. These fibers were correctly oriented and they were either from donor or hybrid origin. The dystrophin-positive fibers remained stable up to 9 months. Possible humoral and cellular immune responses were investigated after grafting. Antibodies directed against dystrophin and/or muscle membrane were developed by 58% of the mice as demonstrated by immunohistochemistry and Western blotting. Despite the presence of these antibodies, dystrophin-positive fibers were still present in grafted muscles 9 months after transplantation. Moreover, the muscles did not show massive infiltration by CD4 cells, CD8 cells, or macrophages, as already described in myoblast allotransplantations. This lack of rejection was attributed to the sequestrated nature of dystrophin after fiber formation. These results indicate that myoblast transplantation leads to fiber formation when immunocompetent but fully histocompatible donors and recipients are used and that dystrophin incompatibility alone is not sufficient to induce an immunological rejection reaction.

Animals↗
PubMed1995

Receptor-mediated gene transfer into human T lymphocytes via binding of DNA/CD3 antibody particles to the CD3 T cell receptor complex.

Retrovirus-mediated gene transfer is currently the method of choice for the transfection of human T lymphocytes for applications in gene therapy. Use of retroviral vectors, however, is hampered by limits on the size of the genetic material to be transferred, the requirement of dividing target cells, and by potential safety questions. Synthetic peptide-enhanced or adenovirus-enhanced receptor-mediated transferrinfection of DNA (SPET and AVET, respectively) is a powerful method for the introduction of genetic material into mammalian cells. Although transferrin has proven to be a useful ligand for gene transfer in many cell types, gene expression in T cells with transferrin/DNA complexes is usually not satisfactory. To improve gene transfer to T cells, antibodies directed against the CD3-T cell receptor complex were tested for their ability to function as ligands for DNA delivery. In T cell lines, up to 50% of the cells expressed a beta-galactosidase reporter gene using anti-CD3 gene transfer complexes. Applying optimized conditions, prestimulated primary peripheral blood lymphocytes were also transfected successfully, although at a lesser efficiency (5%).

Antibodies↗
PubMed1995

Brain metabolic function in older men with chronic essential hypertension.

BACKGROUND AND METHODS. To determine the effects of hypertension on brain function, positron emission tomography (PET) studies using (18F)-2-fluoro-2-deoxy-D-glucose (FDG) were performed on a group of 17 otherwise healthy older hypertensive men (mean age +/- SD = 69 +/- 8 yr) and 25 age- and gender-matched controls. Subjects had medically treated essential hypertension for a minimum of 10 years (range = 10 to 24 yr) with no evidence of end-organ impairment from hypertension by routine clinical screening and by history. All hypertensive and control subjects were determined to be cognitively normal by extensive neuropsychological testing. The hypertensive subjects previously had been reported to have lateral ventricle enlargement and left hemisphere brain atrophy by quantitative MRI. PET data were analyzed using t-tests to look at group differences.

Aged↗
PubMed1995

Glucose kinase-dependent catabolite repression in Staphylococcus xylosus.

By transposon Tn917 mutagenesis, 16 mutants of Staphylococcus xylosus were isolated that showed higher levels of beta-galactosidase activity in the presence of glucose than the wild-type strain. The transposons were found to reside in three adjacent locations in the genome of S. xylosus. The nucleotide sequence of the chromosomal fragment affected by the Tn917 insertions yielded an open reading frame encoding a protein with a size of 328 amino acids with a high level of similarity to glucose kinase from Streptomyces coelicolor. Weaker similarity was also found to bacterial fructokinases and xylose repressors of gram-positive bacteria. The gene was designated glkA. Immediately downstream of glkA, two open reading frames were present whose deduced gene products showed no obvious similarity to known proteins. Measurements of catabolic enzyme activities in the mutant strains grown in the presence or absence of sugars established the pleiotropic nature of the mutations. Besides beta-galactosidase activity, which had been used to detect the mutants, six other tested enzymes were partially relieved from repression by glucose. Reduction of fructose-mediated catabolite repression was observed for some of the enzyme activities. Glucose transport and ATP-dependent phosphorylation of HPr, the phosphocarrier of the phosphoenolpyruvate:carbohydrate phosphotransferase system involved in catabolite repression in gram-positive bacteria, were not affected. The cloned glkA gene fully restored catabolite repression in the mutant strains in trans. Loss of GlkA function is thus responsible for the partial relief from catabolite repression. Glucose kinase activity in the mutants reached about 75% of the wild-type level, indicating the presence of another enzyme in S. xylosus. However, the cloned gene complemented an Escherichia coli strain in glucose kinase. Therefore, the glkA gene encodes a glucose kinase that participates in catabolite repression in S. xylosus.

Amino Acid Sequence↗
PubMed1995

Rhinovirus-mediated endosomal release of transfection complexes.

Endocytosis is an efficient method for transfer of genes into mammalian cells. Incorporation of adenovirus particles into gene transfer complexes greatly enhances gene delivery, probably by the release of endocytosed DNA into the cytoplasm. We report here that two different serotypes of human rhinovirus (HRV), HRV2 and HRV14, are also able to enhance receptor-mediated gene transfer. The effect of several compounds known to inhibit viral infection on HRV2- and HRV14-enhanced transfection was examined. WIN I(s) and WIN IV, two compounds which inhibit viral uncoating, had different effects on HRV2- and HRV14-enhanced gene transfer to NIH 3T3 cells. While HRV14-enhanced gene transfer was severely reduced in the presence of these compounds, virtually no effects were observed when HRV2 was used. The use of antiviral compounds thus allowed transfection of human cells, which are normally lysed rapidly upon infection with HRV. Viral activity could be mimicked by using a peptide derived from the N terminus of VP1 of HRV2. This peptide possesses pH-dependent membrane-disrupting activity and enhances gene transfer to NIH 3T3 and HeLa cells.

3T3 Cells↗
PubMed1995

Generation of high-titer retroviral vectors following receptor-mediated, adenovirus-augmented transfection.

A new procedure is described for the generation of high-titer, helper-free retrovirus vectors employing receptor-mediated, adenovirus-augmented transfection into a standard packaging cell line. Viral titers are increased 30-fold to 100-fold in transiently (> 10(5) infectious units per mL) and stable (> 10(7) infectious units per mL) transfected cells as compared with either CaPO4-mediated transfection or retroviral infection of a packaging cell line. Further, expression of the transduced genes was drastically increased in the transfected cells, but, as expected, there was no difference in transduction efficiency and gene expression in the infected target cells. The increases in viral titers were most likely due to the high number of stable, integrated copies of the vector plasmid DNA in the resulting packaging lines following G418 selection. In addition, experiments generating recombinant retroviruses from non-packaging cell lines are presented. The results suggest that this procedure may be of use to generate high-titer retrovirus vectors in packaging cell lines as well as in primary cells, thus providing a technical basis for in vivo gene transfer upon transplantation of these cells into various organs.

3T3 Cells↗
PubMed1994

Synthesis and anti-HIV activity of thiocholesteryl-coupled phosphodiester antisense oligonucleotides incorporated into immunoliposomes.

Encapsulation of oligonucleotides in antibody-targeted liposomes (immunoliposomes) which bind to target cells permits intracellular delivery of the oligonucleotides. This approach circumvents problems of extracellular degradation by nucleases and poor membrane permeability which free phosphodiester oligonucleotides are subject to, but leaves unresolved the inefficiency of encapsulation of oligonucleotides in liposomes. We have coupled oligonucleotides to cholesterol via a reversible disulfide bond. This modification of oligonucleotides improved their association with immunoliposomes by a factor of about 10 in comparison to unmodified oligonucleotides. The presence of cholesteryl-modified oligonucleotides incorporated in the bilayer of liposomes did not interfere with the coupling of the targeting protein to the liposome surface. Free or cholesterol coupled oligonucleotides associated with liposomes and directed against the tat gene of HIV-1 were tested for inhibition of HIV-1 proliferation in acutely infected cells. We demonstrate that the cholesteryl-modified as well as unmodified oligonucleotides acquire the target specificity of the antibody on the liposome. Their antiviral activity when delivered into cells is sequence-specific. The activity of these modified or unmodified oligonucleotides to inhibit the replication of HIV was the same on an equimolar basis (EC50 around 0.1 microM). Cholesterol coupled oligonucleotides thus offer increased liposome association without loss of antiviral activity.

Antibodies↗
PubMed1994

Gene therapy for B-cell lymphoma in a SCID mouse model using an immunoglobulin-regulated diphtheria toxin gene delivered by a novel adenovirus-polylysine conjugate.

Despite advances in conventional therapy, many lives continue to be lost to common forms of B-cell cancers, including leukemias, lymphomas and multiple myeloma. We propose a novel approach to therapy of such cancers using controlled expression of a diphtheria toxin gene (DT-A) to kill malignant cells. We have previously demonstrated selective killing of various cell types, in vitro and in vivo, by cell-specific, transcriptionally controlled expression of this gene. Organ-specific ablation in otherwise healthy transgenic mice has convincingly demonstrated the exquisite specificity achievable by this technique. In the studies now described, DT-A was delivered in vitro and in vivo using a novel gene delivery system employing DNA physically attached to the exterior of adenovirus. After demonstrating the efficacy of gene delivery to Epstein-Barr virus transformed human B-cells in vitro, in vivo work was performed using a SCID mouse model for B-cell lymphoma, in which protection against tumor was observed. The concepts of tissue-regulated toxin gene therapy, and this novel adenovirus gene delivery system are discussed.

Adenoviridae↗
PubMed1994

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells.

The mucin-type carbohydrate Tn cryptantigen (GalNAc alpha 1-O-Ser/Thr, where GalNAc is N-acetyl-D-galactosamine) is expressed in many carcinomas, in haemopoietic disorders including the Tn syndrome, and on human immunodeficiency virus (HIV) coat glycoproteins, but is not expressed on normal, differentiated cells because of the expression of a Tn-processing galactosyltransferase. Using Jurkat T leukaemic cells which express high levels of Tn antigen due to deficient Tn galactosylation, we have established the Tn antigen-mediated gene transfer and demonstrate the considerable efficiency of this approach. We used poly(L-lysine) conjugates of the monoclonal antibody 1E3 directed against the Tn antigen to deliver the luciferase and beta-galactosidase reporter genes to Jurkat cells by receptor-mediated endocytosis. Addition of unconjugated 1E3 reduced transfection efficiency in a concentration-dependent manner and incubation with free GalNAc abolished DNA transfer completely, indicating that gene delivery is indeed mediated by the Tn antigen. Pre-treatment of Jurkat cells with Vibrio cholerae sialidase, which uncovers additional Tn antigens, resulted in an improvement of gene transfection. Both human and chicken adenovirus particles attached to the DNA/polylysine complex strongly augmented transgene expression. When the beta-galactosidase (lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis, up to 60% of the cells were positive in the cytochemical stain using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) as a chromogenic substrate. The efficiency of the transferrin receptor-mediated DNA uptake into Jurkat cells was comparatively low, although these cells were shown to express considerable amounts of transferrin receptor. We show here that a mucin-type carbohydrate antigen mediates highly efficient DNA uptake by endocytosis into Jurkat T cells. This method represents a 50-fold improvement of Jurkat cell transfection efficiency over other physical gene transfer techniques. Specific gene delivery to primary cancer cells exhibiting Tn epitopes may especially be desirable in immunotherapy protocols.

Adenoviridae↗
PubMed1994

Genetic modification of cells by receptor-mediated adenovirus-augmented gene delivery: a new approach for immunotherapy of cancer.

Most concepts of gene therapy of cancer are based on the generation of an enhanced immune response against the cancer by means of vaccination with gene-modified cancer cells. We have investigated the applicability of a new gene transfer technique which uses the receptor-mediated endocytosis pathway and the endosome disruption activity of adenovirus for the generation of a cancer vaccine consisting of interleukin-2 (IL-2)-transfected, irradiated murine melanoma cells (clone M-3). This technique resulted in very high IL-2 expression (in the range of 30,000 Units IL-2/10(6) cells/24 hrs) in the transfected cells without the need to selection of stably expressing cell clones. We found that this high IL-2 expression of the melanoma cells correlates with high efficacy of the vaccine. Immunization of animals with this vaccine elicits a systemic T-cell-mediated immune response which protects from tumor development after implantation of highly tumorigenic doses of wild-type melanoma cells.

Adenoviridae↗
PubMed1994

Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary human cells in the presence of adenovirus.

Endotoxin (lipopolysaccharide, LPS) is commonly found as a contaminant in plasmid DNA preparations. We demonstrate here that the quantities of LPS typically contaminating DNA preparations can generate a toxicity to primary cells (primary human skin fibroblasts, primary human melanoma cells) in the presence of entry-competent adenovirus particles. Toxicity can be observed with as little as 100 ng/ml free LPS or 100 pg/ml LPS when the LPS is assembled into polylysine/adenovirus complexes. Simple and effective methods of removing the contaminating LPS using either a polymyxin B resin or Triton X-114 extraction are described. Treatment of DNA samples to remove LPS eliminates the toxicity to primary cells.

Adenoviridae↗
PubMed1994

Receptor-mediated gene delivery employing lectin-binding specificity.

Selective targeting of malignant cells will be necessary to implement many of the gene therapy strategies being designed to combat cancer. Targeting can be achieved by transductional or transcriptional approaches. Transductional targeting can be accomplished by exploiting differences in the molecules or receptors expressed on the cell surface of malignant versus normal cells. Given that malignant cells can be distinguished from normal by differences in the expression of cell surface carbohydrates, we hypothesized that transductional targeting would be feasible by molecular conjugate vectors which achieve cell binding by virtue of lectins directed against the cell surface glycocalyx. We have shown that gene transfer can be accomplished by these novel lectin-targeted molecular conjugate vectors and that lectin binding specificities may serve as a means for potential targeting of cancer cells for the purposes of gene therapy.

Animals↗