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https://www.readbyqxmd.com/read/29781215/strap-deficiency-impairs-mouse-embryonic-stem-cells-lineage-commitment-through-cyp26a1-mediated-retinoic-acid-homeostasis
#1
Lin Jin, Chenbei Chang, Kevin M Pawlik, Arunima Datta, Larry M Johnson, Trung Vu, Joseph L Napoli, Pran K Datta
Retinoic acid (RA) signaling is essential for the differentiation of embryonic stem cells (ESCs) and vertebrate development. RA biosynthesis and metabolism are controlled by a series of enzymes, but the molecular regulators of these enzymes remain largely obscure. In this study, we investigated the functional role of the WD-domain protein STRAP (serine threonine kinase receptor-associated protein) in the pluripotency and lineage commitment of murine ESCs. We generated Strap knockout (KO) mouse ESCs and subjected them to spontaneous differentiation...
May 21, 2018: Stem Cells
https://www.readbyqxmd.com/read/29777035/septin-dependent-remodeling-of-cortical-microtubule-drives-cell-reshaping-during-epithelial-wound-healing
#2
Asako Shindo, Anastasia Audrey, Maki Takagishi, Masahide Takahashi, John B Wallingford, Makoto Kinoshita
Wounds in embryos heal rapidly through the contraction of the wound edges. Despite well-recognized significance of the actomyosin purse-string for wound closure, roles for other cytoskeletal components are largely unknown. Here, we report that the septin cytoskeleton cooperates with actomyosin and microtubules to coordinate circumferential contraction of the wound margin and concentric elongation of wound-proximal cells in Xenopus laevis embryos. Microtubules reoriented radially, forming bundles along lateral cell cortices in elongating wound-proximal cells...
May 18, 2018: Journal of Cell Science
https://www.readbyqxmd.com/read/29773519/the-neurosteroid-allopregnanolone-sulfate-inhibits-na-v-1-3-%C3%AE-subunit-containing-voltage-gated-sodium-channels-expressed-in-xenopus-oocytes
#3
Takafumi Horishita, Nobuyuki Yanagihara, Susumu Ueno, Dan Okura, Reiko Horishita, Tomoko Minami, Yuichi Ogata, Yuka Sudo, Yasuhito Uezono, Takashi Kawasaki
The neurosteroid allopregnanolone has potent analgesic effects, and its potential use for neuropathic pain is supported by recent reports. However, the analgesic mechanisms are obscure. The voltage-gated sodium channels (Nav ) α subunit Nav 1.3 is thought to play an essential role in neuropathic pain. Here, we report the effects of allopregnanolone sulfate (APAS) on sodium currents (INa ) in Xenopus oocytes expressing Nav 1.3 with β1 or β3 subunits. APAS suppressed INa of Nav 1.3 with β1 and β3 in a concentration-dependent manner (IC50 values; 75 and 26 μmol/L)...
March 30, 2018: Journal of Pharmacological Sciences
https://www.readbyqxmd.com/read/29772211/synthesis-folding-structure-and-activity-of-a-predicted-peptide-from-the-sea-anemone-oulactis-sp-with-an-shkt-fold
#4
Bankala Krishnarjuna, Jessica Villegas-Moreno, Michela L Mitchell, Agota Csoti, Steve Peigneur, Carlos Amero, Michael W Pennington, Jan Tytgat, Gyorgy Panyi, Raymond S Norton
Sea anemone venom is rich in bioactive compounds, including peptides containing multiple disulfide bridges. In a transcriptomic study on Oulactis sp., we identified the putative 36-residue peptide, OspTx2b, which is an isoform of the KV channel blocker OspTx2a (Sunanda P et al. (2018) Identification, chemical synthesis, structure and function of a new KV 1 channel blocking peptide from Oulactis sp. Peptide Science, in press). As OspTx2b contains a ShK/BgK-like cysteine framework, with high amino acid sequence similarity to BgK, we were interested to investigate its structure and function...
May 14, 2018: Toxicon: Official Journal of the International Society on Toxinology
https://www.readbyqxmd.com/read/29772022/binding-of-sulphonylureas-to-plasma-proteins-a-katp-channel-perspective
#5
Peter Proks, Holger Kramer, Elizabeth Haythorne, Frances M Ashcroft
Sulphonylurea drugs stimulate insulin secretion from pancreatic β-cells primarily by inhibiting ATP sensitive potassium (KATP) channels in the β-cell membrane. The effective sulphonylurea concentration at its site of action is significantly attenuated by binding to serum albumin, which makes it difficult to compare in vitro and in vivo data. We therefore measured the ability of gliclazide and glibenclamide to inhibit KATP channels and stimulate insulin secretion in the presence of serum albumin. We used this data, together with estimates of free drug concentrations from binding studies, to predict the extent of sulphonylurea inhibition of KATP channels at therapeutic concentrations in vivo...
2018: PloS One
https://www.readbyqxmd.com/read/29769720/rspo2-inhibition-of-rnf43-and-znrf3-governs-limb-development-independently-of-lgr4-5-6
#6
Emmanuelle Szenker-Ravi, Umut Altunoglu, Marc Leushacke, Célia Bosso-Lefèvre, Muznah Khatoo, Hong Thi Tran, Thomas Naert, Rivka Noelanders, Amin Hajamohideen, Claire Beneteau, Sergio B de Sousa, Birsen Karaman, Xenia Latypova, Seher Başaran, Esra Börklü Yücel, Thong Teck Tan, Lena Vlaeminck, Shalini S Nayak, Anju Shukla, Katta Mohan Girisha, Cédric Le Caignec, Natalia Soshnikova, Zehra Oya Uyguner, Kris Vleminckx, Nick Barker, Hülya Kayserili, Bruno Reversade
The four R-spondin secreted ligands (RSPO1-RSPO4) act via their cognate LGR4, LGR5 and LGR6 receptors to amplify WNT signalling1-3 . Here we report an allelic series of recessive RSPO2 mutations in humans that cause tetra-amelia syndrome, which is characterized by lung aplasia and a total absence of the four limbs. Functional studies revealed impaired binding to the LGR4/5/6 receptors and the RNF43 and ZNRF3 transmembrane ligases, and reduced WNT potentiation, which correlated with allele severity. Unexpectedly, however, the triple and ubiquitous knockout of Lgr4, Lgr5 and Lgr6 in mice did not recapitulate the known Rspo2 or Rspo3 loss-of-function phenotypes...
May 16, 2018: Nature
https://www.readbyqxmd.com/read/29769401/microinjection-of-mrnas-and-oligonucleotides
#7
Sally A Moody
Microinjecting lineage tracers into a single blastomere in the normal, intact embryo identifies the repertoire of cell types derived from it. In order to reveal the full developmental potential of that blastomere or identify the mechanisms by which its fate is determined, one needs to modify its gene expression under controlled experimental conditions. One method by which this is easily accomplished in Xenopus is by microinjecting synthetic mRNAs or antisense oligonucleotides into an identified blastomere to target altered gene expression specifically to its lineage...
May 16, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29769400/calculating-the-degradation-rate-of-individual-proteins-using-xenopus-extract-systems
#8
Gary S McDowell, Anna Philpott
The Xenopus extract system has been used extensively as a simple, quick, and robust method for assessing the stability of proteins against proteasomal degradation. In this protocol, methods are provided for assessing the half-life of in vitro translated radiolabeled proteins using Xenopus egg or embryo extracts.
May 16, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29769399/dissecting-and-culturing-animal-cap-explants
#9
Kevin S Dingwell, James C Smith
The animal cap explant is a simple but adaptable tool available to developmental biologists. The use of animal cap explants in demonstrating the presence of mesoderm-inducting activity in the Xenopus embryo vegetal pole is one of many elegant examples of their worth. Animal caps respond to a range of growth factors (e.g., Wnts, FGF, TGF-β), making them especially useful for studying signal transduction pathways and gene regulatory networks. Explants are also suitable for examining cell behavior and have provided key insights into the molecular mechanisms controlling vertebrate morphogenesis...
May 16, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29769398/cleavage-blastomere-deletion-and-transplantation-to-test-cell-fate-commitment-in-xenopus
#10
Sally A Moody
Fate maps identify the precursors of an organ, and tracing the members of a blastomere lineage over time shows how its descendants come to populate that organ. The fates of the individual blastomeres of the two- to 32-cell Xenopus embryo have been fully mapped to reveal which cells are the major contributors to various cell types, tissues, and organs. However, because these fate maps were produced in the normal embryo, they do not reveal whether a precursor blastomere is competent to give rise to additional tissues or is already committed to its fate-mapped repertoire of descendants...
May 16, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29769397/larval-thymectomy-of-xenopus-laevis
#11
Sara Mashoof, Breanna Breaux, Michael F Criscitiello
In jawed vertebrates from sharks to mammals, the thymus is the primary (or central) lymphoid tissue where T cells develop and mature. The particular stromal cell types, cytokine environment, and tissue organization in the thymus are essential for V(D)J recombination, positive selection for major histocompatibility complex recognition, and negative selection against self-peptide recognition of most αβ T cells. The thymectomy operation on Xenopus tadpole larva described here creates a T-cell-deficient model suitable for many immunology studies...
May 16, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29769396/bulk-electroporation-mediated-gene-transfer-into-xenopus-tadpole-brain
#12
Cristina Sáenz de Miera, Ethan Parr, Robert J Denver
In vivo gene transfer is a powerful tool for investigating protein function and gene regulation in living organisms. Delivery of plasmid DNA to the brain of Xenopus tadpoles by bulk electroporation-mediated (EM) gene transfer can be used to study the effects of ectopic gene expression on development, physiology, and behavior. It can also be used to mark cells for lineage tracing, investigate the in vivo function of gene regulatory elements when linked to a reporter gene, and introduce mutations into the genome of transfected cells, among other applications...
May 16, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29769395/methods-for-investigating-the-larval-period-and-metamorphosis-in-xenopus
#13
Daniel R Buchholz, Yun-Bo Shi
Anuran metamorphosis resembles postembryonic development in mammals, a period around birth when many organs/tissues mature into their adult form as circulating thyroid and stress hormone levels are high. Unlike uterus-enclosed mammalian embryos, tadpoles develop externally and undergo the dramatic changes of hormone-dependent development totally independent of maternal influence, making them a valuable model in which to study vertebrate postembryonic organ development and maturation. Various protocols have been developed and/or adapted for studying metamorphosis in Xenopus laevis and X...
May 16, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29769394/analysis-of-cell-fate-commitment-in-xenopus-embryos
#14
Sally A Moody
The fates of individual cleavage-stage blastomeres and of groups of cells at the blastula through gastrula stages of Xenopus embryos have been mapped in great detail. These studies identified the major contributors of the three germ layers as well as a variety of tissues and organs and several specific cell types. One can use these fate maps to test the commitment of single cells or groups of cells to produce their normal repertoire of descendants, to identify the genes that regulate fate commitment, and to modulate the levels of gene expression in specific lineages to determine gene function in a variety of developmental processes...
May 16, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29769393/tracing-central-nervous-system-axon-regeneration-in-xenopus
#15
Kurt M Gibbs, Ben G Szaro
Axonal tracing allows visualizing connectivity between neurons, providing useful information about structure, neuronal location, and function of the nervous system. Identifying regenerating axons and their neuron cell bodies present the particular challenges of labeling the projections of interest while unambiguously demonstrating regrowth of those axons that have been damaged. In the developing brain, an additional labeling challenge arises, as new connections are being made throughout the duration of an experiment...
May 16, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29769392/cleavage-blastomere-explant-culture-in-xenopus
#16
Sally A Moody
The individual blastomeres of Xenopus two- to 32-cell embryos have been fate mapped. This work identified the precursors of most of the embryonic cell types, tissues and organs; however, the maps do not reveal the cell interactions or signaling pathways that are required for establishing cell fates. This protocol describes an explant culture approach for culturing blastomeres in isolation to test whether a cell's fate has been determined. Cleavage blastomeres can be cultured in a simple salt medium without added factors because they contain intracellular yolk platelets, which provide an intrinsic energy source...
May 16, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29769391/infrared-laser-mediated-gene-induction-at-the-single-cell-level-in-the-regenerating-tail-of-xenopus-laevis-tadpoles
#17
Riho Hasugata, Shinichi Hayashi, Aiko Kawasumi-Kita, Joe Sakamoto, Yasuhiro Kamei, Hitoshi Yokoyama
We describe a precise and reproducible gene-induction method in the amphibian, Xenopus laevis Tetrapod amphibians are excellent models for studying the mechanisms of three-dimensional organ regeneration because they have an exceptionally high regenerative ability. However, spatial and temporal manipulation of gene expression has been difficult in amphibians, hindering studies on the molecular mechanisms of organ regeneration. Recently, however, development of a Xenopus transgenic system with a heat-shock-inducible gene has enabled the manipulation of specific genes...
May 16, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29769390/cell-transplantation-as-a-method-to-investigate-spinal-cord-regeneration-in-regenerative-and-nonregenerative-xenopus-stages
#18
Emilio E Méndez-Olivos, Juan Larraín
Mammals are not capable of regenerating their central nervous system (CNS); anamniotes, however, can regenerate in response to injury. The mechanisms that explain the different regenerative capabilities include: (i) extrinsic mechanisms that consider the cellular environment and extracellular matrix composition, (ii) intrinsic factors implicating the presence or absence of genetic programs that promote axon regeneration, and (iii) the presence or absence of neural stem and progenitors cells (NSPCs) that allow neurogenesis...
May 16, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29769388/lineage-tracing-and-fate-mapping-in-xenopus-embryos
#19
Sally A Moody
Fate mapping approaches reveal what types of cells, tissues, and organs are derived from specific embryonic cells. Classical fate maps were made by microscopic techniques using embryos comprising small numbers of transparent cells. More complex and opaque embryos require use of a vital or lipophilic dye that labels small groups of cells. Intracellular injection of a lineage tracer that labels the injected cell and all of its descendants can be used to mark a single cell in Xenopus embryos, whose large cells are easy to microinject and usually cleave in regular patterns...
May 16, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29769387/assessing-ubiquitylation-of-individual-proteins-using-xenopus-extract-systems
#20
Gary S McDowell, Anna Philpott
Xenopus extract systems have been used to study ubiquitylation of proteins, and to uncover some of the fundamental processes of the ubiquitylation pathway itself. They provide a simple, quick, and robust method for studying ubiquitylation. In this protocol, methods are provided for studying protein ubiquitylation using Xenopus egg or embryo extracts and in vitro radiolabeled proteins. These methods also enable examination of whether proteins undergo noncanonical ubiquitylation, through modification of the protein by covalent linkage to ubiquitin through residues other than lysine, such as cysteine, serine, and threonine...
May 16, 2018: Cold Spring Harbor Protocols
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