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D Sackett

Publications and source records attributed to D Sackett.

At least 19 recordsLinked to original sources
PubMed2005

Quantitative method to study the network formation of endothelial cells in response to tumor angiogenic factors.

To study the network formation of endothelial cells (ECs) in an extracellular matrix (ECM) environment, we have devised an EC aggregation-type model based on a diffusion limited cluster aggregation model (DLCA), where clusters of particles diffuse and stick together upon contact. We use this model to quantify EC differentiation into cord-like structures by comparing experimental and simulation data. Approximations made with the DLCA model, when combined with experimental kinetics and cell concentration results, not only allow us to quantify cell differentiation by a pseudo diffusion coefficient, but also measure the effects of tumor angiogenic factors (TAFs) on the formation of cord-like structures by ECs. We have tested our model by using an in vitro assay, where we record EC aggregation by analysing time-lapse images that provide us with the evolution of the fractal dimension measure through time. We performed these experiments for various cell concentrations and TAFs (e.g. EVG, FGF-b, and VEGF). During the first six hours of an experiment, ECs aggregate quickly. The value of the measured fractal dimension decreases with time until reaching an asymptotic value that depends solely on the EC concentration. In contrast, the kinetics depend on the nature of TAFs. The experimental and simulation results correlate with each other in regards to the fractal dimension and kinetics, allowing us to quantify the influence of each TAF by a pseudo diffusion coefficient. We have shown that the shape, kinetic aggregation, and fractal dimension of the EC aggregates fit into an in vitro model capable of reproducing the first stage of angiogenesis. We conclude that the DLCA model, combined with experimental results, is a highly effective assay for the quantification of the kinetics and network characteristics of ECs embedded in ECM proteins. Finally, we present a new method that can be used for studying the effect of angiogenic drugs in in vitro assays.

Angiogenesis Inducing Agents↗
PubMed2000

A common pharmacophore for epothilone and taxanes: molecular basis for drug resistance conferred by tubulin mutations in human cancer cells.

The epothilones are naturally occurring antimitotic drugs that share with the taxanes a similar mechanism of action without apparent structural similarity. Although photoaffinity labeling and electron crystallographic studies have identified the taxane-binding site on beta-tubulin, similar data are not available for epothilones. To identify tubulin residues important for epothilone binding, we have isolated two epothilone-resistant human ovarian carcinoma sublines derived in a single-step selection with epothilone A or B. These epothilone-resistant sublines exhibit impaired epothilone- and taxane-driven tubulin polymerization caused by acquired beta-tubulin mutations (beta274(Thr-->Ile) and beta282(Arg-->Gln)) located in the atomic model of alphabeta-tubulin near the taxane-binding site. Using molecular modeling, we investigated the conformational behavior of epothilone, which led to the identification of a common pharmacophore shared by taxanes and epothilones. Although two binding modes for the epothilones were predicted, one mode was identified as the preferred epothilone conformation as indicated by the activity of a potent pyridine-epothilone analogue. In addition, the structure-activity relationships of multiple taxanes and epothilones in the tubulin mutant cells can be fully explained by the model presented here, verifying its predictive value. Finally, these pharmacophore and activity data from mutant cells were used to model the tubulin binding of sarcodictyins, a distinct class of microtubule stabilizers, which in contrast to taxanes and the epothilones interact preferentially with the mutant tubulins. The unification of taxane, epothilone, and sarcodictyin chemistries in a single pharmacophore provides a framework to study drug-tubulin interactions that should assist in the rational design of agents targeting tubulin.

Alkaloids↗
PubMed2000

P21-dependent g(1)arrest with downregulation of cyclin D1 and upregulation of cyclin E by the histone deacetylase inhibitor FR901228.

Depsipeptide, FR901228, a novel cyclic peptide inhibitor of histone deacetylase with a unique cytotoxicity profile is currently in phase I clinical trials. Here we demonstrate that, in addition to G2/M arrest, FR901228 causes G1 arrest with Rb hypophosphorylation. In vitro kinase assays demonstrated no direct inhibition of CDK activity, however, an inhibition was observed in CDKs extracted from cells exposed to FR901228. Cyclin D1 protein disappeared between 6 and 12 hours after treatment with FR901228, whereas cyclin E was upregulated. While it did not induce wt p53, FR901228 did induce p21(WAF1/CIP1)in a p53-independent manner. Cell clones lacking p21 were not arrested in G1 phase, but continued DNA synthesis and were arrested in G2/M phase following FR901228 treatment. Finally, FR901228 blunted ERK-2/MAPK activation by EGF whereas early signal transduction events remained intact since overall cellular tyrosine phosphorylation after EGF stimulation was unaffected. Thus, FR901228, while not directly inhibiting kinase activity, causes cyclin D1 downregulation and a p53-independent p21 induction, leading to inhibition of CDK and dephosphorylation of Rb resulting in growth arrest in the early G1 phase. In contrast to the G1 arrest, the G2/M arrest is p21-independent, but is associated with significant cytotoxicity.

Anti-Bacterial Agents↗
PubMed1999

D-dimer for the exclusion of venous thromboembolism: comparison of a new automated latex particle immunoassay (MDA D-dimer) with an established enzyme-linked fluorescent assay (VIDAS D-dimer).

The use of D-dimer tests to exclude venous thromboembolism is an important advance in clinical practice and also has economic benefits. Ideally the test should be objective and a test that could be run on the routine coagulometer would obviate the need for additional investment in alternative hardware. A new automated latex particle immunoassay (MDA D-dimer) that can be run on a routine coagulometer was compared with a well established enzyme linked fluorescent assay (VIDAS D-dimer) on the basis of their ability to exclude venous thromboembolism. The assays were compared in 49 patients presenting to the emergency department with clinically suspected deep vein thrombosis or pulmonary embolism. After objective diagnostic imaging, 20 patients were confirmed to have venous thromboembolism. There was strong agreement between the assays in individual patients. Using a cut-off of 500 micrograms/l, both tests had a sensitivity of 100% and therefore a negative predictive value of 100%, however the MDA test would have spared more patients (20% vs. 12%) from further testing if a negative D-dimer was used to rule-out the diagnosis. It was concluded that a rapid, objective latex D-dimer test run on a routine coagulometer (MDA D-dimer) can be used to exclude the diagnosis of venous thromboembolism.

Enzyme-Linked Immunosorbent Assay↗
PubMed1997

Taxanes.

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Animals↗
PubMed1996

Isolation of a biologically active soluble human interferon-alpha receptor-GST fusion protein expressed in Escherichia coli.

The cDNA encoding the extracellular domain of the human interferon-alpha (IFN-alpha) receptor (Uzé, G., Lutfalla, G., and Gresser, I. Cell 1990;60:225-234) lacking the signal peptide has been expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The fusion protein represented 12% of total bacterial proteins and was found exclusively within cytoplasmic inclusion bodies. Inclusion body material was completely solubilized by 8 M urea; 20% solubilization was achieved by cell lysis in the presence of 0.45% cholamidopropyl dimethylammoniol-propane sulfonate and 1% Triton X-100. The soluble fusion protein was purified by gel filtration and affinity chromatography. Overall recovery of affinity purified fusion protein was approximately 100-200 micrograms/liter of cell culture. The affinity purified and refolded fusion protein exhibited the expected amino terminal sequence and M(r) of 68,000 on reduced sodium dodecylsulfate gel electrophoresis. The protein reacted with antibodies specific for the cloned IFN-alpha receptor and inhibited the antiviral and antiproliferative activities of recombinant IFN-alpha B. We have demonstrated that the fusion protein binds to IFN-alpha B and competes with the cell surface receptor for binding to this IFN-alpha species.

Antiviral Agents↗
PubMed1996

Human interferon-alpha receptor: identification of the region involved in binding to interferon-alpha B.

Three polypeptides comprising amino acids 1-102, 93-260, and 261-410 of the extracellular domain of the human interferon-alpha receptor HuIFN-alpha R (Uzé, G., Lutfalla, G., and Gresser, I. Cell 1990; 60:225-234) have been expressed in Escherichia coli. The polypeptides were sequestered within bacterial inclusion bodies. Inclusion body material was solubilized by 8 M urea, and the polypeptides were purified by gel filtration or histidine tag-based affinity chromatography. Overall recovery of each purified and refolded polypeptide was approximately 0.5-0.8 mg/liter of cell culture. The polypeptides migrated as homogeneous monomers of 12 kDa, 22 kDa, and 17 kDa, respectively on reduced sodium dodecylsulfate polyacrylamide gel electrophoresis. The polypeptide fragments corresponding to amino acids 1-102, and 93-260 of the extracellular domain of HuIFN-alpha R lacked the ability to bind to IFN-alpha B and to inhibit its biologic activities. The polypeptide fragment corresponding to amino acids 261-410 of the receptor molecule inhibited the antiproliferative activity of IFN-alpha B and competed with the Daudi cell surface receptor for binding to this IFN-alpha species.

Antiviral Agents↗