Other search tools

About this data

The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.

If you have any questions or comments, please contact us.

Results: 1 to 10 of 19

Publication Record

Connections

Chemical Screening Using Cell-Free Egg Extract.
Broadus MR, Lee E
(2018) Cold Spring Harb Protoc 2018:
MeSH Terms: Animals, Cell-Free System, Drug Evaluation, Preclinical, Ovum, Xenopus laevis
Show Abstract · Added April 8, 2018
Most drug screening methods use purified proteins, cultured cells, and/or small model organisms such as , zebrafish, flies, or nematodes. These systems have proven successes in drug discovery, but they also have weaknesses. Although purified cellular components allow for identification of compounds with activity against specific targets, such systems lack the complex biological interactions present in cellular and organismal screens. In vivo systems overcome these weaknesses, but the lack of cellular permeability, efflux by cellular pumps, and/or toxicity can be major limitations. egg extract, a concentrated and biologically active cytosol, can potentially overcome these weaknesses. Drug interactions occur in a near-physiological milieu, thereby functioning in a "truer" endogenous manner than purified components. Also, egg extract is a cell-free system that lacks intact plasma membranes that could restrict drug access to potential targets. Finally, egg extract is readily manipulated at the protein level: Proteins are easily depleted or added to the system, an important feature for analyzing drug effects in disease states. Thus, egg extract offers an attractive media for screening drugs that merges strengths of both in vitro and in vivo systems.
© 2018 Cold Spring Harbor Laboratory Press.
0 Communities
1 Members
0 Resources
5 MeSH Terms
Small-molecule high-throughput screening utilizing Xenopus egg extract.
Broadus MR, Yew PR, Hann SR, Lee E
(2015) Methods Mol Biol 1263: 63-73
MeSH Terms: Animals, Cell-Free System, Drug Evaluation, Preclinical, Gene Expression, High-Throughput Screening Assays, In Vitro Techniques, Luciferases, Ovum, Recombinant Fusion Proteins, Small Molecule Libraries, Xenopus
Show Abstract · Added November 2, 2015
Screens for small-molecule modulators of biological pathways typically utilize cultured cell lines, purified proteins, or, recently, model organisms (e.g., zebrafish, Drosophila, C. elegans). Herein, we describe a method for using Xenopus laevis egg extract, a biologically active and highly tractable cell-free system that recapitulates a legion of complex chemical reactions found in intact cells. Specifically, we focus on the use of a luciferase-based fusion system to identify small-molecule modulators that affect protein turnover.
0 Communities
1 Members
0 Resources
11 MeSH Terms
Reconstitution Of β-catenin degradation in Xenopus egg extract.
Chen TW, Broadus MR, Huppert SS, Lee E
(2014) J Vis Exp :
MeSH Terms: Animals, Female, Ovum, Xenopus laevis, beta Catenin
Show Abstract · Added November 2, 2015
Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways(1-3). Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, β-catenin. Two different methods are described to assess β-catenin protein degradation in Xenopus egg extract. One method is visually informative ([(35)S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess β-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of β-catenin degradation.
0 Communities
1 Members
0 Resources
5 MeSH Terms
Molecular and functional characterization of Anopheles gambiae inward rectifier potassium (Kir1) channels: a novel role in egg production.
Raphemot R, Estévez-Lao TY, Rouhier MF, Piermarini PM, Denton JS, Hillyer JF
(2014) Insect Biochem Mol Biol 51: 10-9
MeSH Terms: Amino Acid Sequence, Animals, Anopheles, Base Sequence, Female, Molecular Sequence Data, Oocytes, Ovary, Ovum, Potassium Channels, Inwardly Rectifying, Pupa, RNA Interference, Xenopus laevis
Show Abstract · Added February 5, 2016
Inward rectifier potassium (Kir) channels play essential roles in regulating diverse physiological processes. Although Kir channels are encoded in mosquito genomes, their functions remain largely unknown. In this study, we identified the members of the Anopheles gambiae Kir gene family and began to investigate their function. Notably, we sequenced the A. gambiae Kir1 (AgKir1) gene and showed that it encodes all the canonical features of a Kir channel: an ion pore that is composed of a pore helix and a selectivity filter, two transmembrane domains that flank the ion pore, and the so-called G-loop. Heterologous expression of AgKir1 in Xenopus oocytes revealed that this gene encodes a functional, barium-sensitive Kir channel. Quantitative RT-PCR experiments then showed that relative AgKir1 mRNA levels are highest in the pupal stage, and that AgKir1 mRNA is enriched in the adult ovaries. Gene silencing of AgKir1 by RNA interference did not affect the survival of female mosquitoes following a blood meal, but decreased their egg output. These data provide evidence for a new role of Kir channels in mosquito fecundity, and further validates them as promising molecular targets for the development of a new class of mosquitocides to be used in vector control.
Copyright © 2014 Elsevier Ltd. All rights reserved.
0 Communities
1 Members
0 Resources
13 MeSH Terms
Neurosensory perception of environmental cues modulates sperm motility critical for fertilization.
McKnight K, Hoang HD, Prasain JK, Brown N, Vibbert J, Hollister KA, Moore R, Ragains JR, Reese J, Miller MA
(2014) Science 344: 754-7
MeSH Terms: Animals, Caenorhabditis elegans, Caenorhabditis elegans Proteins, Environmental Exposure, Female, Fertilization, Male, Neurons, Afferent, Neurosecretory Systems, Oocytes, Ovum, Perception, Pheromones, Prostaglandin-Endoperoxide Synthases, Prostaglandins, Sperm Motility, Spermatozoa, Transforming Growth Factor beta
Show Abstract · Added May 19, 2014
Environmental exposures affect gamete function and fertility, but the mechanisms are poorly understood. Here, we show that pheromones sensed by ciliated neurons in the Caenorhabditis elegans nose alter the lipid microenvironment within the oviduct, thereby affecting sperm motility. In favorable environments, pheromone-responsive sensory neurons secrete a transforming growth factor-β ligand called DAF-7, which acts as a neuroendocrine factor that stimulates prostaglandin-endoperoxide synthase [cyclooxygenase (Cox)]-independent prostaglandin synthesis in the ovary. Oocytes secrete F-class prostaglandins that guide sperm toward them. These prostaglandins are also synthesized in Cox knockout mice, raising the possibility that similar mechanisms exist in other animals. Our data indicate that environmental cues perceived by the female nervous system affect sperm function.
Copyright © 2014, American Association for the Advancement of Science.
0 Communities
1 Members
0 Resources
18 MeSH Terms
Preventing the transmission of pathogenic mitochondrial DNA mutations: Can we achieve long-term benefits from germ-line gene transfer?
Samuels DC, Wonnapinij P, Chinnery PF
(2013) Hum Reprod 28: 554-9
MeSH Terms: Animals, Chimera, DNA, Mitochondrial, Extrachromosomal Inheritance, Female, Gene Transfer Techniques, Genetic Diseases, Inborn, Genetic Therapy, Germ Cells, Humans, Models, Genetic, Mutation, Ovum, Reproductive Techniques, Assisted, United Kingdom
Show Abstract · Added December 12, 2013
Mitochondrial medicine is one of the few areas of genetic disease where germ-line transfer is being actively pursued as a treatment option. All of the germ-line transfer methods currently under development involve some carry-over of the maternal mitochondrial DNA (mtDNA) heteroplasmy, potentially delivering the pathogenic mutation to the offspring. Rapid changes in mtDNA heteroplasmy have been observed within a single generation, and so any 'leakage' of mutant mtDNA could lead to mtDNA disease in future generations, compromising the reproductive health of the first generation, and leading to repeated interventions in subsequent generations. To determine whether this is a real concern, we developed a model of mtDNA heteroplasmy inheritance by studying 87 mother-child pairs, and predicted the likely outcome of different levels of 'mutant mtDNA leakage' on subsequent maternal generations. This showed that, for a clinical threshold of 60%, reducing the proportion of mutant mtDNA to <5% dramatically reduces the chance of disease recurrence in subsequent generations, but transmitting >5% mutant mtDNA was associated with a significant chance of disease recurrence. Mutations with a lower clinical threshold were associated with a higher risk of recurrence. Our findings provide reassurance that, at least from an mtDNA perspective, methods currently under development have the potential to effectively eradicate pathogenic mtDNA mutations from subsequent generations.
0 Communities
1 Members
0 Resources
15 MeSH Terms
Screening for small molecule inhibitors of embryonic pathways: sometimes you gotta crack a few eggs.
Hang BI, Thorne CA, Robbins DJ, Huppert SS, Lee LA, Lee E
(2012) Bioorg Med Chem 20: 1869-77
MeSH Terms: Animals, Drug Evaluation, Preclinical, Hedgehog Proteins, High-Throughput Screening Assays, Ovum, Receptors, Notch, Signal Transduction, Small Molecule Libraries, Wnt Signaling Pathway, Xenopus laevis
Show Abstract · Added March 5, 2014
Extract prepared from Xenopus eggs represents a cell-free system that has been shown to recapitulate a multitude of cellular processes, including cell cycle regulation, DNA replication/repair, and cytoskeletal dynamics. In addition, this system has been used to successfully reconstitute the Wnt pathway. Xenopus egg extract, which can be biochemically manipulated, offers an ideal medium in which small molecule screening can be performed in near native milieu. Thus, the use of Xenopus egg extract for small molecule screening represents an ideal bridge between targeted and phenotypic screening approaches. This review focuses on the use of this system for small molecules modulators of major signal transduction pathways (Notch, Hedgehog, and Wnt) that are critical for the development of the early Xenopus embryo. We describe the properties of Xenopus egg extract and our own high throughput screen for small molecules that modulate the Wnt pathway using this cell-free system. We propose that Xenopus egg extract could similarly be adapted for screening for modulators of the Notch and Hedgehog pathways.
Copyright © 2011 Elsevier Ltd. All rights reserved.
1 Communities
3 Members
0 Resources
10 MeSH Terms
Fast microtubule dynamics in meiotic spindles measured by single molecule imaging: evidence that the spindle environment does not stabilize microtubules.
Needleman DJ, Groen A, Ohi R, Maresca T, Mirny L, Mitchison T
(2010) Mol Biol Cell 21: 323-33
MeSH Terms: Animals, Biopolymers, Cell Extracts, Cyclopropanes, Kinetics, Meiosis, Microtubules, Molecular Imaging, Ovum, Photobleaching, Pyridines, Thiazoles, Time Factors, Tubulin, Xenopus laevis
Show Abstract · Added March 5, 2014
Metaphase spindles are steady-state ensembles of microtubules that turn over rapidly and slide poleward in some systems. Since the discovery of dynamic instability in the mid-1980s, models for spindle morphogenesis have proposed that microtubules are stabilized by the spindle environment. We used single molecule imaging to measure tubulin turnover in spindles, and nonspindle assemblies, in Xenopus laevis egg extracts. We observed many events where tubulin molecules spend only a few seconds in polymer and thus are difficult to reconcile with standard models of polymerization dynamics. Our data can be quantitatively explained by a simple, phenomenological model-with only one adjustable parameter-in which the growing and shrinking of microtubule ends is approximated as a biased random walk. Microtubule turnover kinetics did not vary with position in the spindle and were the same in spindles and nonspindle ensembles nucleated by Tetrahymena pellicles. These results argue that the high density of microtubules in spindles compared with bulk cytoplasm is caused by local enhancement of nucleation and not by local stabilization. It follows that the key to understanding spindle morphogenesis will be to elucidate how nucleation is spatially controlled.
0 Communities
1 Members
0 Resources
15 MeSH Terms
LRP6 transduces a canonical Wnt signal independently of Axin degradation by inhibiting GSK3's phosphorylation of beta-catenin.
Cselenyi CS, Jernigan KK, Tahinci E, Thorne CA, Lee LA, Lee E
(2008) Proc Natl Acad Sci U S A 105: 8032-7
MeSH Terms: Adaptor Proteins, Signal Transducing, Animals, Axin Protein, Cattle, Cell Extracts, Dishevelled Proteins, Embryo, Nonmammalian, Glycogen Synthase Kinase 3, Intracellular Space, Low Density Lipoprotein Receptor-Related Protein-6, Mice, Ovum, Phosphoproteins, Phosphorylation, Proteasome Endopeptidase Complex, Protein Processing, Post-Translational, Protein Structure, Tertiary, Receptors, LDL, Recombinant Proteins, Repressor Proteins, Signal Transduction, Thermodynamics, Ubiquitin, Wnt Proteins, Xenopus, Xenopus Proteins, beta Catenin
Show Abstract · Added March 5, 2014
Wnt/beta-catenin signaling controls various cell fates in metazoan development and is misregulated in several cancers and developmental disorders. Binding of a Wnt ligand to its transmembrane coreceptors inhibits phosphorylation and degradation of the transcriptional coactivator beta-catenin, which then translocates to the nucleus to regulate target gene expression. To understand how Wnt signaling prevents beta-catenin degradation, we focused on the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6), which is required for signal transduction and is sufficient to activate Wnt signaling when overexpressed. LRP6 has been proposed to stabilize beta-catenin by stimulating degradation of Axin, a scaffold protein required for beta-catenin degradation. In certain systems, however, Wnt-mediated Axin turnover is not detected until after beta-catenin has been stabilized. Thus, LRP6 may also signal through a mechanism distinct from Axin degradation. To establish a biochemically tractable system to test this hypothesis, we expressed and purified the LRP6 intracellular domain from bacteria and show that it promotes beta-catenin stabilization and Axin degradation in Xenopus egg extract. Using an Axin mutant that does not degrade in response to LRP6, we demonstrate that LRP6 can stabilize beta-catenin in the absence of Axin turnover. Through experiments in egg extract and reconstitution with purified proteins, we identify a mechanism whereby LRP6 stabilizes beta-catenin independently of Axin degradation by directly inhibiting GSK3's phosphorylation of beta-catenin.
0 Communities
2 Members
0 Resources
27 MeSH Terms
Cell-free extract systems and the cytoskeleton: preparation of biochemical experiments for transmission electron microscopy.
Coughlin M, Brieher WM, Ohi R
(2007) Methods Mol Biol 369: 199-212
MeSH Terms: Actins, Animals, Biotin, Cell-Free System, Cytoskeleton, Female, Fixatives, Listeria monocytogenes, Microscopy, Electron, Transmission, Ovum, Plastic Embedding, Spindle Apparatus, Streptavidin, Xenopus laevis
Show Abstract · Added March 5, 2014
Cell-free systems can be used to reconstitute complex actin or microtubule-based phenomena. For example, a number of extracts support actin-dependent propulsion of Listeria monocytogenes, whereas Xenopus laevis extracts support formation of a microtubule-based meiotic spindle. Working in vitro opens these complex processes to biochemical dissection. Here, we describe methods to view these in vitro preparations by thin-section electron microscopy.
0 Communities
1 Members
0 Resources
14 MeSH Terms