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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.02.033 Byline: Yi Ying Wang (a), Amanda Charlesworth (b)(c), Shannon M. Byrd (e), Robert Gregerson (f), Melanie C. MacNicol (b)(c), Angus M. MacNicol (a)(b)(d) Abstract: Cell cycle progression during oocyte maturation requires the strict temporal regulation of maternal mRNA translation. The intrinsic basis of this temporal control has not been fully elucidated but appears to involve distinct mRNA 3' UTR regulatory elements. In this study, we identify a novel translational control sequence (TCS) that exerts repression of target mRNAs in immature oocytes of the frog, Xenopus laevis, and can direct early cytoplasmic polyadenylation and translational activation during oocyte maturation. The TCS is functionally distinct from the previously characterized Musashi/polyadenylation response element (PRE) and the cytoplasmic polyadenylation element (CPE). We report that TCS elements exert translational repression in both the Wee1 mRNA 3' UTR and the pericentriolar material-1 (Pcm-1) mRNA 3' UTR in immature oocytes. During oocyte maturation, TCS function directs the early translational activation of the Pcm-1 mRNA. By contrast, we demonstrate that CPE sequences flanking the TCS elements in the Wee1 3' UTR suppress the ability of the TCS to direct early translational activation. Our results indicate that a functional hierarchy exists between these distinct 3' UTR regulatory elements to control the timing of maternal mRNA translational activation during oocyte maturation. Author Affiliation: (a) Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Slot 814, 4301 W. Markham St., Little Rock, AR 72205, USA (b) Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Slot 814, 4301 W. Markham St., Little Rock, AR 72205, USA (c) The Center for Translational Neuroscience, University of Arkansas for Medical Sciences, Slot 814, 4301 W. Markham St., Little Rock, AR 72205, USA (d) The Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Slot 814, 4301 W. Markham St., Little Rock, AR 72205, USA (e) Committee on Developmental Biology, The University of Chicago, Chicago, IL, USA (f) Science Division, Lyon College, Batesville, AR, USA Article History: Received 5 June 2007; Revised 14 February 2008; Accepted 15 February 2008

The Musashi family of mRNA translational regulators controls both physiological and pathological stem cell self-renewal primarily by repressing target mRNAs that promote differentiation. In response to differentiation cues, Musashi can switch from a repressor to an activator of target mRNA translation. However, the molecular events that distinguish Musashi-mediated translational activation from repression are not understood. We have previously reported that Musashi function is required for the maturation of Xenopus oocytes and specifically for translational activation of specific dormant maternal mRNAs. Here, we employed MS to identify cellular factors necessary for Musashi-dependent mRNA translational activation. We report that Musashi1 needs to associate with the embryonic poly(A)-binding protein (ePABP) or the canonical somatic cell poly(A)-binding protein PABPC1 for activation of Musashi target mRNA translation. Co-immunoprecipitation studies demonstrated an increased Musashi1 interaction with ePABP during oocyte maturation. Attenuation of endogenous ePABP activity severely compromised Musashi function, preventing downstream signaling and blocking oocyte maturation. Ectopic expression of either ePABP or PABPC1 restored Musashi-dependent mRNA translational activation and maturation of ePABP-attenuated oocytes. Consistent with these Xenopus findings, PABPC1 remained associated with Musashi under conditions of Musashi target mRNA de-repression and translation during mammalian stem cell differentiation. Because association of Musashi1 with poly(A)-binding proteins has previously been implicated only in repression of Musashi target mRNAs, our findings reveal novel context-dependent roles for the interaction of Musashi with poly(A)-binding protein family members in response to extracellular cues that control cell fate. [ABSTRACT FROM AUTHOR]

Background In recent years, 3D printing technology has become inexpensive and simple enough that any lab can own and use one of these printers. New method We explored the potential use of 3D printers for quickly and easily producing in vitro slice chambers for patch clamp electrophysiology. Slice chambers were produced using five available plastics: ABS, PLA, Nylon 618, Nylon 680, and T-glase. These “lab-made” chambers were also made using stereolithography through a professional printing service (Shapeways). This study measured intrinsic membrane properties of neurons in the brain stem pedunculopontine nucleus (PPN) and layer V pyramidal neurons in retrosplenial cortex. Results Nylon 680 and T-glase significantly hyperpolarized PPN neurons. ABS increased input resistance, decreased action potential amplitude, and increased firing frequency in pyramidal cortical neurons. To test long term exposure to each plastic, human neuroblastoma SHSY5Y cell cultures were exposed to each plastic for 1 week. ABS decreased cell counts while Nylon 618 and Shapeways plastics eliminated cells. Primary mouse pituitary cultures were also tested for 24-h exposure. ABS decreased cell counts while Nylon 618 and Shapeways plastics dramatically decreased cell counts. Comparison to existing methods Chambers can be quickly and inexpensively printed in the lab. ABS, PLA, Nylon 680, and T-glase plastics would suffice for many experiments instead of commercially produced slice chambers. Conclusions While these technologies are still in their infancy, they represent a powerful addition to the lab environment. With careful selection of print material, slice chambers can be quickly and inexpensively manufactured in the lab. [ABSTRACT FROM AUTHOR]

Cell cycle re-entry during vertebrate oocyte maturation is mediated through translational activation of select target mRNAs, culminating in the activation of mitogen-activated protein kinase and cyclin B/cyclin-dependent kinase (CDK) signaling. The temporal order of targeted mRNA translation is crucial for cell cycle progression and is determined by the timing of activation of distinct mRNA-binding proteins. We have previously shown in oocytes from Xenopus laevis that the mRNA-binding protein Musashi targets translational activation of early class mRNAs including the mRNA encoding the Mos proto-oncogene. However, the molecular mechanism by which Musashi function is activated is unknown. We report here that activation of Musashi1 is mediated by Ringo/CDK signaling, revealing a novel role for early Ringo/CDK function. Interestingly, Musashi1 activation is subsequently sustained through mitogen-activated protein kinase signaling, the downstream effector of Mos mRNA translation, thus establishing a positive feedback loop to amplify Musashi function. The identified regulatory sites are present in mammalian Musashi proteins, and our data suggest that phosphorylation may represent an evolutionarily conserved mechanism to control Musashi-dependent target mRNA translation. [ABSTRACT FROM AUTHOR]

In response to hormonal stimulation quiescent 3T3-L1 preadipocyte cells reenter the cell cycle and undergo a mitotic expansion phase prior to terminal differentiation. The cell cycle regulatory proteins p130 and p107 undergo dramatic changes in protein levels within 24 h of differentiation. The role of these proteins in regulating adipocyte mitotic clonal expansion and/or differentiation are unclear. It has recently been demonstrated that adipocyte proliferation can be uncoupled from adipocyte differentiation through the use of the pharmacological MEK inhibitor PD98059 or the tyrosine phosphatase inhibitor, sodium vanadate. We examined the expression of p130 and p107 in stimulated 3T3-L1 cells in the presence of either PD98059, U0126 or sodium vanadate. While inhibition of MEK blocked proliferation, the cells underwent differentiation normally. In contrast, vanadate blocked differentiation without affecting proliferation. Inhibition of MEK did not affect the increase in p107 expression in stimulated cells indicating that induction of p107 is independent of MAP kinase signaling. Vanadate treatment caused a significant delay in p107 expression in the first 24 h following stimulation. Under these conditions, p130 expression was relatively unchanged. Our results indicate that a rapid increase in p107 expression correlates with a commitment to undergo adipocyte differentiation. The data further suggest that the rapid induction of p107 is not required for cellular proliferation during the mitotic clonal expansion phase. Taken together, these findings provide correlative data that implicate p107 in the terminal differentiation, but not proliferation, of quiescent preadipocytes following hormonal stimulation. [Copyright &y& Elsevier]

Breast cancer remains one of the leading cancers among women. Cancer stem cells (CSCs) are tumor-initiating cells which drive progression, metastasis, and reoccurrence of the disease. CSCs are resistant to conventional chemo- and radio-therapies and their ability to survive such treatment enables tumor reestablishment. Metastasis is the main cause of mortality in women with breast cancer, thus advances in treatment will depend on therapeutic strategies targeting CSCs. Salinomycin (SAL) is a naturally occurring polyether ionophore antibiotic known for its anticancer activity towards several types of tumor cells. In the present work, a library of 17 C1-single and C1/C20-double modified SAL analogs was screened to identify compounds with improved activity against breast CSCs. Six single- and two double-modified analogs were more potent (IC 50 range of 1.1 ± 0.1–1.4 ± 0.2 µM) toward the breast cancer cell line MDA-MB-231 compared to SAL (IC 50 of 4.9 ± 1.6 µM). Double-modified compound 17 was found to be more efficacious than SAL against the majority of cancer cell lines in the NCI-60 Human Tumor Cell Line Panel. Compound 17 was more potent than SAL in inhibiting cell migration and cell renewal properties of MDA-MB-231 cells, as well as inducing selective loss of the CD44+/CD24 /low stem-cell-like subpopulation in both monolayer (2D) and organoid (3D) culture. The present findings highlight the therapeutic potential of SAL analogs towards breast CSCs and identify select compounds that merit further study and clinical development. [Display omitted] • Single- and double-modified salinomycin analogs were more potent towards MDA-MB-231 breast cancer cells than salinomycin • Double modified analog 17 induced DNA fragmentation and apoptotic cell death in MDA-MB-231 cell monolayer and organoids • Analog 17 reduced colony formation potential and migration of MDA-MB-231 cells to the greater extent than salinomycin • Analog 17 more effectively than salinomycin reduced CD44+/CD24- cancer stem-like cells in monolayer and organoid cultures [ABSTRACT FROM AUTHOR]