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E Wagner

Publications and source records attributed to E Wagner.

At least 145 records · Page 8Linked to original sources
PubMed1996

Activation of the complement system by synthetic DNA complexes: a potential barrier for intravenous gene delivery.

We have examined the complement-activating properties of synthetic cationic molecules and their complexes with DNA. Commonly used gene delivery vehicles include complexes of DNA with polylysine of various chain lengths, transferrin-polylysine, a fifth-generation poly(amidoamine) (PAMAM) dendrimer, poly(ethyleneimine), and several cationic lipids (DOTAP, DC-Chol/DOPE, DOGS/DOPE, and DOTMA/DOPE). These agents activate the complement system to varying extents. Strong complement activation is seen with long-chain polylysines, the dendrimer, poly(ethyleneimine), and DOGS (half-maximal at about 3 microM amine content in the assay used). Compared to these compounds, the other cationic lipids (in liposome formulations) are weak activators of the complement system (half-maximal approximately 50-100 microM positive charge in assay). Complement activation by polylysine is strongly dependent on the chain length. Short-chain oligolysines are comparable to cationic lipids in their activation of complement. Incubation of these compounds with DNA to form complexes reduces complement activation in virtually all cases. The degree of complement activation by DNA complexes is strongly dependent on the ratio of polycation and DNA (expressed as the charge ratio) for polylysine, dendrimer, poly(ethyleneimine), and DOGS. To a lesser degree, charge ratio also influences complement activation by monovalent cationic lipid-DNA complexes. For polylysine-DNA complexes, complement activation can be considerably reduced by modifying the surface of preformed DNA complexes with polyethyleneglycol (half-maximal approximately 20 microM amine content). The data suggests that, by appropriate formulation of DNA complexes, complement activation can be minimized or even avoided. These findings should facilitate the search for DNA complex formulations appropriate for reproducible intravenous gene delivery.

Animals↗
PubMed1996

Inactivation of ribonucleotide reductase by (E)-2'-fluoromethylene-2'-deoxycytidine 5'-diphosphate: a paradigm for nucleotide mechanism-based inhibitors.

Ribonucleotide reductase (RDPR) from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two homodimeric subunits: R1 and R2. (E)- and (Z)-2'-fluoromethylene-2'-deoxycytidine 5'-diphosphate (FMCDP) are time dependent inactivators of this protein, with approximately 1.5 equiv being sufficient for complete loss of catalytic activity. Inactivation results from loss of the essential tyrosyl radical on R2 and alkylation of R1. Studies using electron spin resonance spectroscopy reveal that tyrosyl radical loss is accompanied by formation of a new, substrate-based radical. Experiments using [6'-14C]-(E)-FMCDP and [5-3H]-(E)-FMCDP reveal that alkylation of R1 is accompanied by release of 0.5 equiv of cytosine and 1.4 equiv of fluoride ion. When R1 is denatured subsequent to inactivation, approximately 1 equiv of label per R1 is observed only in studies carried out with [14C]FMCDP. Under these same conditions with [3H]FMCDP, 1.5 equiv of radiolabel is detected as cytosine. Inactivation of R1 thus results from alkylation by the sugar moiety of FMCDP. While studies to isolate the alkylated amino acid on R1 were unsuccessful, studies using a variety of site-directed mutants of R1 (C462S, C225S, C754/759S, C439S, and E441Q) indicate that E441 or possibly C439 is the modified residue. Inactivation is accompanied by rapid formation of a new chromophore with a lambda max at 334 nm. Dithiothreitol does not protect the enzyme against inactivation by FMCDP, although it does prevent chromophore formation. Two possible mechanisms are proposed to accommodate these experimental observations.

Alkylation↗
PubMed1996

Extrachromosomal recombination occurs efficiently in cells defective in various DNA repair systems.

A series of different frameshift mutations of a firefly luciferase reporter plasmid was created so that no activity was obtained when they were transfected into mammalian cells. Co-transfection of these constructs with short fragments of the original sequence resulted in luciferase activity in different cell lines (A-549, NIH 3T3 and Jurkat). The level of this activity was dependent on the length of the fragment, regardless of cell line examined. Two different transfection techniques (lipofection and adenovirus-enhanced gene transfer) gave similar results. It was shown by polymerase chain reaction that expression of detectable luciferase required recombination of the transfected molecules. Cells with defined defects in DNA repair pathways were examined for their ability to perform this extrachromosomal recombination. Cells lacking normal Ku p80, (ADP-ribosyl)transferase, MLH1 or XP-C were all capable of restoring expression to the frameshifted constructs. Given the pivotal roles of the above molecules in the pathways of DNA repair, it seems that this recombination derives from a different activity.

3T3 Cells↗
PubMed1996

HLA-B27 determination using serological methods. A comparison of enzyme immunoassay and a microlymphocytotoxic test with flow cytometry and a molecular biological assay.

Typing for HLA-B27 is routinely performed in patients with seronegative spondarthritides. Besides the microlymphocytotoxic test (MLCT), other serological techniques have been developed such as enzyme immunoassays (EIA) using serum or plasma as a source for the determination of soluble HLA-B27 (sHLA-B27) and flow cytometric (FC) methods. The aim of the present study was to check the accuracy and reliability of the EIA for sHLA-B27 in comparison to the MLCT using antibodies against HLA-B27 and cross-reacting specificities (CRS), as well as an FC method and a molecular biological method. Any discrepant results should be typed with the MLCT using a complete panel of anti-HLA-class I antibodies, with FC and with a molecular biological technique. The EIA should also be repeated in those patients, using serum and plasma from a new venipuncture. In 81 patients with rheumatic disorders, the EIA and the MLCT using antibodies against HLA-B27 and CRS were performed. Based on the MLCT with a complete panel of anti-HLA-class I antibodies as a standard, discrepant test results were obtained for 9 out of 81 patients with the MLCT using antibodies against HLA-B27 and CRS and with the EIA. The following wrong results occurred: in the MLCT with anti-HLA-B27 and CRS, there were two false-negative results; in the EIA there were four false-negative and one false-positive results; one sample was undeterminable. In comparison with the MLCT, including the complete panel of HLA-class I antibodies, as well as with a molecular biological technique, typing with FC showed a complete concordance. Our investigations demonstrated that for routine typing for HLA-B27 the MLCT cannot be replaced by EIA because of a significant number of mistypings. The MLCT performed only with antibodies against HLA-B27 and CRS may also lead to typing errors. No errors were detected using flow cytometry. If only serological methods can be performed in a laboratory a combination of flow cytometry and MLCT could therefore enhance the safety of HLA-B27 typing.

Adult↗
PubMed1996

Predictors of chronic benzodiazepine use in a health maintenance organization sample.

While expert recommendations caution against long-term benzodiazepine use in the elderly, survey data suggest increasing benzodiazepine use with age. Computerized pharmacy records of staff-model HMO were used to examine benzodiazepine prescribing. Six-month prevalence of benzodiazepine use (2.8%) and prevalence of continued use (0.7%) were lower than earlier reports. Prevalence was higher in women and increased steadily with age. Among 7012 patients beginning benzodiazepine treatment, duration of use increased with patient age, prescription by a psychiatrist (vs. primary care or medical/surgical specialist), use of higher-potency drugs (lorazepam, and alprazolem, clonazepam) and larger number of pills in the initial prescription. Individual physicians varied significantly in drug choice, initial prescription size, and likelihood of chronic use. Among 200 patients treated in primary care, the physician-recorded indication for prescription was anxiety or depression in 27%, insomnia in 20%, and pain symptoms in 38%. These findings indicate a gap between benzodiazepine efficacy research and current clinical practice.

Adolescent↗
PubMed1996

Fractal analysis and imaging of the proximal nephron cell.

Cells of the S1 proximal renal tubule were examined to determine whether their peculiar shapes are a result of certain constructs of fractal mathematics. Morphometric measurements of the cell perimeter were made at several levels of cell height by measuring the intercellular boundaries that appear on electron micrographs of tubule cross sections. When the measurements were made over a range of scale lengths, the fractal dimension, D, of the cell perimeter was found to increase from 1.3 near the cell apex to 1.78 near the cell base. The length of scale was found to range between 8 and 0.4 micron and to represent the approximate dimensions of actual cell processes. Fractal patterns that conformed to the measured parameters were then constructed from a fractal generator composed of budlike formations that originated near the cell apex and that increased in number and decreased in size with cell depth according to a fractal scaling. It was found that the fractal rule of keeping a constant relative scale could be maintained between budding processes but, to obtain patterns that resemble biological structure, the processes must be positioned randomly on the cell periphery. It is shown that when the relative sizes of the buds decrease exponentially and their numbers increase geometrically, the perimeter can grow to the correct length without overlap. This suggests that patterns of the cell periphery corresponding to different levels of cell height obey a law of scale but occur randomly in a way that increases to high fractal dimension or near plane-filling values at the cell base. The fractal patterns that correspond to the measured fractal dimensions can be assembled into a three-dimensional model that closely resembles the known shape of the proximal tubule cell.

Animals↗
PubMed1996

Glycerol enhancement of ligand-polylysine/DNA transfection.

Primary human fibroblasts and a series of cell lines (A549, BNL CL.2, H225, NIH 3T3 and Rat-1) are efficiently transfected by using positively charged complexes of plasmid DNA and transferrin-polylysine or polylysine in the presence of glycerol (1 molar to 1.8 molar, depending on the cell type). An increase in gene expression of up to several-hundredfold (compared to complexes without glycerol) is obtained if the transfection mixture is incubated with the cells for 3-4 h at 37 degrees C. This simple method has been used for transient expression of luciferase, beta-galactosidase and interleukin-2, and also for the generation of stably transfected cells.

Animals↗
PubMed1996

Developmental and cell cycle regulation of alfalfa nucMs1, a plant homolog of the yeast Nsr1 and mammalian nucleolin.

We report here the isolation and characterization of the nucMs1 alfalfa cDNA, whose predicted amino acid sequence structurally resembles the yeast Nsr1 protein and animal nucleolins. These proteins consist of an N-terminal acidic domain, centrally located RNA recognition motifs (RRMs), and a C-terminal glycine- and arginine-rich domain. In comparison with animal nucleolins that contain four RRMs, NucMs1 more closely resembles the yeast Nsr1 protein, which contains only two RRMs. A NucMs1 C-terminal peptide antibody specifically recognized a 95-kD nucleolar protein in alfalfa cells that changed its localization in a cell cycle-dependent manner. The nucMs1 transcript and p95nucMs1 protein levels correlated with cell proliferation, and nucMs1 gene expression was found to be induced in the G1 phase upon mitogenic stimulation of G0-arrested leaf cells. In situ hybridization analysis of different alfalfa organs during various developmental stages showed that nucMs1 gene expression is highest in root meristematic cells, but it is also found in other meristematic cells of the plant body. nucMs1 expression is tightly linked to cell proliferation but does not depend on a particular cell cycle phase. No nucMs1 expression was observed in cells that had exited the cell cycle and were undergoing differentiation or polar growth, indicating that nucMs1 may not be necessary for processes other than cell proliferation.

Animals↗
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

Elicitation of a systemic and protective anti-melanoma immune response by an IL-2-based vaccine. Assessment of critical cellular and molecular parameters.

We have established a model for the immunologic rejection of melanoma cells. Using a receptor-mediated, adenovirus-augmented gene delivery system (transferrinfection) we have shown that, upon transfection with an IL-2 gene construct, MHC class I+/class II- murine M-3 cells lose their tumorigenicity in both athymic and euthymic mice. More importantly, we found that these melanoma cells, which produce high levels of IL-2, can be used to induce a long-lasting anti-tumor immune response in syngeneic euthymic DBA/2 mice but not in athymic animals. This immune response, which can also be elicited by coadministration of nonmodified, irradiated M-3 cells and IL-2-transduced fibroblasts, results in the rejection of a subsequent challenge with M-3 cells or, in the elimination of preexisting M-3 cancer cell deposits. We found that transfer of T cell-enriched, but not of T cell-depleted, splenocytes from immunized mice conferred protection against M-3 cells, but not against unrelated KLN 205 cancer cells. Transfer of either CD4+ or CD8+ T cells led to only partial protection against challenge with wild-type M-3 cells. Our further observations that T cell-enriched, but not T cell-depleted splenocytes of immunized animals are capable of tumor-specific lytic activity and that this activity resides in the CD8+ cell population are compatible with the assumption that MHC class I-restricted T cell cytotoxicity is a biologically relevant effector mechanism in this model. That other mechanisms also contribute to melanoma cell destruction is evidenced by the presence of large numbers of macrophages and granulocytes in addition to T cells at the challenge sites of immunized mice.

Animals↗
PubMed1995

Regulation of the tissue factor promoter in endothelial cells. Binding of NF kappa B-, AP-1-, and Sp1-like transcription factors.

Tissue factor is up-regulated on endothelial cells and monocytes in response to cytokines and endotoxin and is the main trigger of the extrinsic pathway of the coagulation cascade. We have isolated the porcine tissue factor gene and studied the regulation of the promoter, which has not been investigated previously in endothelial cells. Comparison of the promoter sequences with the respective human and murine genes reveals short stretches of homology, which encompass potential binding sites for AP-1, NF kappa B, and Sp1 transcription factors. Using DNase I footprinting, we detect binding of nuclear factors to these promoter elements. Transfection experiments demonstrate that a 300-base pair fragment containing the conserved elements can mediate induced transcription and that the NF kappa B-like element is essential. In accordance, electrophoretic mobility shift assays show a strong increase in the binding of factors to the NF kappa B-like site following induction. We further provide evidence that RelA (p65), c-Rel, and possibly novel polypeptides bind to the tissue factor NF kappa B element. In addition, we show constitutive binding of members of the Fos/Jun and Sp1 families to the AP-1 and Sp1 sites, respectively. We propose a concerted action of AP-1-, NF kappa B-, and Sp1-like factors in transcription from the tissue factor promoter in endothelial cells.

Animals↗
PubMed1995

Utility of pharmacodynamic measures for assessing the oral bioavailability of peptides. 1. Administration of recombinant salmon calcitonin in rats.

Salmon calcitonin (sCT) is a therapeutic peptide used in the treatment of Paget's Disease, postmenopausal osteoporosis, and hypercalcemia due to malignancy. In this study, recombinant sCT (rsCT) was administered intravenously (iv), subcutaneously (sc), and intraduodenally (id.) in rats to evaluate pharmacodynamic (PD) response as a measure of rsCT bioavailability (F) and to test the feasibility of delivering rsCT orally. rsCT pharmacokinetics were linear throughout the range of iv and sc doses studied. Following sc administration, F ranged from 11.2% to 23.1% and was linear. The absorption of rsCT after id. administration was low (0.022%); however, a significant lowering of serum calcium concentrations was observed. Serum calcium lowering was nonlinear and saturable after sc administration with the minimum dose required for maximum calcium lowering (Dmin/max) equal to 10.2 ng and a maximal response of 426.8 mg min/dL. Using Dmin/max as the reference dose, absolute Fs were recalculated using PD response after id. administration of 1 and 2 mg of rsCT and were 0.040% and 0.029%, respectively. Substantial overestimates of F were obtained when the reference dose was not properly selected. While the absorption of rsCT was low, the significant lowering of serum calcium levels suggests that oral delivery of sCT is feasible. The results of these studies also suggest that PD response is useful in assessing the oral bioavailability of peptides; however, when PD response is saturable, as is the case for rsCT, the reference dose should be carefully selected in order to avoid overestimates of F.

Administration, Oral↗
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

Cost-effectiveness comparisons using "real world" randomized trials: the case of new antidepressant drugs.

While randomized clinical trials remain the "gold standard" for comparisons of treatment efficacy, conventional randomized trials may not reflect the relative effectiveness of treatments under "real world" conditions. Observational data more closely reflect the conditions of actual practice, but are often limited in clinical detail and subject to bias in selection of treatments. The authors use available data on the cost-effectiveness of new antidepressant drugs to illustrate the limitations of these methods. Modifications to the traditional randomized trial may allow more accurate assessment of effectiveness in everyday practice. The authors describe the design of such a "real world" randomized trial comparing newer antidepressants with older alternatives.

Antidepressive Agents↗