Edelfosine-induced metabolic changes in cancer cells that precede the overproduction of reactive oxygen species and apoptosis

Background: Metabolic flux profiling based on the analysis of distribution of stable isotope tracer in metabolites is an important method widely used in cancer research to understand the regulation of cell metabolism and elaborate new therapeutic strategies. Recently, we developed software Isodyn, which extends the methodology of kinetic modeling to the analysis of isotopic isomer distribution for the evaluation of cellular metabolic flux profile under relevant conditions. This tool can be applied to reveal the metabolic effect of proapoptotic drug edelfosine in leukemia Jurkat cell line, uncovering the mechanisms of induction of apoptosis in cancer cells. Results: The study of 13C distribution of Jukat cells exposed to low edelfosine concentration, which induces apoptosis in ¿5% of cells, revealed metabolic changes previous to the development of apoptotic program. Specifically, it was found that low dose of edelfosine stimulates the TCA cycle. These metabolic perturbations were coupled with an increase of nucleic acid synthesis de novo, which indicates acceleration of biosynthetic and reparative processes. The further increase of the TCA cycle fluxes, when higher doses of drug applied, eventually enhance reactive oxygen species (ROS) production and trigger apoptotic program. Conclusion: The application of Isodyn to the analysis of mechanism of edelfosine-induced apoptosis revealed primary drug-induced metabolic changes, which are important for the subsequent initiation of apoptotic program. Initiation of such metabolic changes could be exploited in anticancer therapy

Activity and regulation by growth factors of calmodulin-dependent protein kinase III (elongation factor 2-kinase) in human breast cancer

Calmodulin-dependent protein kinase III (CaM kinase III, elongation factor-2 kinase) is a unique member of the Ca2+/CaM-dependent protein kinase family. Activation of CaM kinase III leads to the selective phosphorylation of elongation factor 2 (eEF-2) and transient inhibition of protein synthesis. Recent cloning and sequencing of CaM kinase III revealed that this enzyme represents a new superfamily of protein kinases. The activity of CaM kinase III is selectively activated in proliferating cells; inhibition of the kinase blocked cells in G0/G1-S and decreased viability. To determine the significance of CaM kinase III in breast cancer, we measured the activity of the kinase in human breast cancer cell lines as well as in fresh surgical specimens. The specific activity of CaM kinase III in human breast cancer cell lines was equal to or greater than that seen in a variety of cell lines with similar rates of proliferation. The specific activity of CaM kinase III was markedly increased in human breast tumour specimens compared with that of normal adjacent breast tissue. The activity of this enzyme was regulated by breast cancer mitogens. In serum-deprived MDA-MB-231 cells, the combination of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) stimulated cell proliferation and activated CaM kinase III to activities observed in the presence of 10% serum. Inhibition of enzyme activity blocked cell proliferation induced by growth factors. In MCF-7 cells separated by fluorescence-activated cell sorting, CaM kinase III was increased in S-phase over that of other phases of the cell cycle. In summary, the activity of Ca2+/CaM-dependent protein kinase III is controlled by breast cancer mitogens and appears to be constitutively activated in human breast cancer. These results suggest that CaM kinase III may contribute an important link between growth factor/receptor interactions, protein synthesis and the induction of cellular proliferation in human breast cancer. © 1999 Cancer Research Campaig

An inverse approach to determine complex modulus gradient of field-aged asphalt mixtures

This study develops a new mechanical-based method to determine the complex modulus and modulus gradient of field-aged asphalt mixtures using the direct tension test. Due to the non-uniform aging nature of the field cores, the mechanical responses must be measured at different depths. Meanwhile, the monotonic load is not applied at the neutral axis of the field core specimen due to the modulus gradient, the tensile part of the strain is used and should be separated from the measurement because of the eccentric loading. The modulus gradient parameters, the location of the neutral axis, and the stress distribution are first obtained using the elastic formulas for a series of loading times. Then the complex modulus is determined using the Laplace transform and the elastic–viscoelastic correspondence principle. An inverse approach and iteration are then proposed by using the pseudo strain to accurately calculate the modulus gradient parameters after the relaxation modulus and reference modulus are determined

Noninvasive Monitoring of Placenta-Specific Transgene Expression by Bioluminescence Imaging

BACKGROUND: Placental dysfunction underlies numerous complications of pregnancy. A major obstacle to understanding the roles of potential mediators of placental pathology has been the absence of suitable methods for tissue-specific gene manipulation and sensitive assays for studying gene functions in the placentas of intact animals. We describe a sensitive and noninvasive method of repetitively tracking placenta-specific gene expression throughout pregnancy using lentivirus-mediated transduction of optical reporter genes in mouse blastocysts. METHODOLOGY/PRINCIPAL FINDINGS: Zona-free blastocysts were incubated with lentivirus expressing firefly luciferase (Fluc) and Tomato fluorescent fusion protein for trophectoderm-specific infection and transplanted into day 3 pseudopregnant recipients (GD3). Animals were examined for Fluc expression by live bioluminescence imaging (BLI) at different points during pregnancy, and the placentas were examined for tomato expression in different cell types on GD18. In another set of experiments, blastocysts with maximum photon fluxes in the range of 2.0E+4 to 6.0E+4 p/s/cm(2)/sr were transferred. Fluc expression was detectable in all surrogate dams by day 5 of pregnancy by live imaging, and the signal increased dramatically thereafter each day until GD12, reaching a peak at GD16 and maintaining that level through GD18. All of the placentas, but none of the fetuses, analyzed on GD18 by BLI showed different degrees of Fluc expression. However, only placentas of dams transferred with selected blastocysts showed uniform photon distribution with no significant variability of photon intensity among placentas of the same litter. Tomato expression in the placentas was limited to only trophoblast cell lineages. CONCLUSIONS/SIGNIFICANCE: These results, for the first time, demonstrate the feasibility of selecting lentivirally-transduced blastocysts for uniform gene expression in all placentas of the same litter and early detection and quantitative analysis of gene expression throughout pregnancy by live BLI. This method may be useful for a wide range of applications involving trophoblast-specific gene manipulations in utero

Incorporating gene co-expression network in identification of cancer prognosis markers

<p>Abstract</p> <p>Background</p> <p>Extensive biomedical studies have shown that clinical and environmental risk factors may not have sufficient predictive power for cancer prognosis. The development of high-throughput profiling technologies makes it possible to survey the whole genome and search for genomic markers with predictive power. Many existing studies assume the interchangeability of gene effects and ignore the coordination among them.</p> <p>Results</p> <p>We adopt the weighted co-expression network to describe the interplay among genes. Although there are several different ways of defining gene networks, the weighted co-expression network may be preferred because of its computational simplicity, satisfactory empirical performance, and because it does not demand additional biological experiments. For cancer prognosis studies with gene expression measurements, we propose a new marker selection method that can properly incorporate the network connectivity of genes. We analyze six prognosis studies on breast cancer and lymphoma. We find that the proposed approach can identify genes that are significantly different from those using alternatives. We search published literature and find that genes identified using the proposed approach are biologically meaningful. In addition, they have better prediction performance and reproducibility than genes identified using alternatives.</p> <p>Conclusions</p> <p>The network contains important information on the functionality of genes. Incorporating the network structure can improve cancer marker identification.</p

Identifying Regulators for EAG1 Channels with a Novel Electrophysiology and Tryptophan Fluorescence Based Screen

Ether-à-go-go (EAG) channels are expressed throughout the central nervous system and are also crucial regulators of cell cycle and tumor progression. The large intracellular amino- and carboxy- terminal domains of EAG1 each share similarity with known ligand binding motifs in other proteins, yet EAG1 channels have no known regulatory ligands.Here we screened a library of small biologically relevant molecules against EAG1 channels with a novel two-pronged screen to identify channel regulators. In one arm of the screen we used electrophysiology to assess the functional effects of the library compounds on full-length EAG1 channels. In an orthogonal arm, we used tryptophan fluorescence to screen for binding of the library compounds to the isolated C-terminal region.Several compounds from the flavonoid, indole and benzofuran chemical families emerged as binding partners and/or regulators of EAG1 channels. The two-prong screen can aid ligand and drug discovery for ligand-binding domains of other ion channels

Black hole growth in the early Universe is self-regulated and largely hidden from view

The formation of the first massive objects in the infant Universe remains impossible to observe directly and yet it sets the stage for the subsequent evolution of galaxies. While some black holes with masses > billion solar masses? have been detected in luminous quasars less than one billion years after the Big Bang, these individual extreme objects have limited utility in constraining the channels of formation of the earliest black holes. The initial conditions of black hole seed properties are quickly erased during the growth process. From deep, optimally stacked, archival X-ray observations, we measure the amount of black hole growth in z=6-8 galaxies (0.7-1 billion years after the Big Bang). Our results imply that black holes grow in tandem with their hosts throughout cosmic history, starting from the earliest times. We find that most copiously accreting black holes at these epochs are buried in significant amounts of gas and dust that absorb most radiation except for the highest energy X-rays. This suggests that black holes grow significantly more than previously thought during these early bursts, and due to obscuration they do not contribute to the re-ionization of the Universe with their ultraviolet emission.Comment: Nature, in pres

A Computational Method for Prediction of Excretory Proteins and Application to Identification of Gastric Cancer Markers in Urine

A novel computational method for prediction of proteins excreted into urine is presented. The method is based on the identification of a list of distinguishing features between proteins found in the urine of healthy people and proteins deemed not to be urine excretory. These features are used to train a classifier to distinguish the two classes of proteins. When used in conjunction with information of which proteins are differentially expressed in diseased tissues of a specific type versus control tissues, this method can be used to predict potential urine markers for the disease. Here we report the detailed algorithm of this method and an application to identification of urine markers for gastric cancer. The performance of the trained classifier on 163 proteins was experimentally validated using antibody arrays, achieving >80% true positive rate. By applying the classifier on differentially expressed genes in gastric cancer vs normal gastric tissues, it was found that endothelial lipase (EL) was substantially suppressed in the urine samples of 21 gastric cancer patients versus 21 healthy individuals. Overall, we have demonstrated that our predictor for urine excretory proteins is highly effective and could potentially serve as a powerful tool in searches for disease biomarkers in urine in general

Search for rare quark-annihilation decays, B --> Ds(*) Phi

We report on searches for B- --> Ds- Phi and B- --> Ds*- Phi. In the context of the Standard Model, these decays are expected to be highly suppressed since they proceed through annihilation of the b and u-bar quarks in the B- meson. Our results are based on 234 million Upsilon(4S) --> B Bbar decays collected with the BABAR detector at SLAC. We find no evidence for these decays, and we set Bayesian 90% confidence level upper limits on the branching fractions BF(B- --> Ds- Phi) Ds*- Phi)<1.2x10^(-5). These results are consistent with Standard Model expectations.Comment: 8 pages, 3 postscript figues, submitted to Phys. Rev. D (Rapid Communications