Characterization of Novel Paternal ncRNAs at the Plagl1 Locus, Including Hymai, Predicted to Interact with Regulators of Active Chromatin

Genomic imprinting is a complex epigenetic mechanism of transcriptional control that utilizes DNA methylation and histone modifications to bring about parent-of-origin specific monoallelic expression in mammals. Genes subject to imprinting are often organised in clusters associated with large non-coding RNAs (ncRNAs), some of which have cis-regulatory functions. Here we have undertaken a detailed allelic expression analysis of an imprinted domain on mouse proximal chromosome 10 comprising the paternally expressed Plagl1 gene. We identified three novel Plagl1 transcripts, only one of which contains protein-coding exons. In addition, we characterised two unspliced ncRNAs, Hymai, the mouse orthologue of HYMAI, and Plagl1it (Plagl1 intronic transcript), a transcript located in intron 5 of Plagl1. Imprinted expression of these novel ncRNAs requires DNMT3L-mediated maternal DNA methylation, which is also indispensable for establishing the correct chromatin profile at the Plagl1 DMR. Significantly, the two ncRNAs are retained in the nucleus, consistent with a potential regulatory function at the imprinted domain. Analysis with catRAPID, a protein-ncRNA association prediction algorithm, suggests that Hymai and Plagl1it RNAs both have potentially high affinity for Trithorax chromatin regulators. The two ncRNAs could therefore help to protect the paternal allele from DNA methylation by attracting Trithorax proteins that mediate H3 lysine-4 methylation

Tocolytic effect of a selective FP receptor antagonist in rodent models reveals an innovative approach to the treatment of preterm labor

<p>Abstract</p> <p>Background</p> <p>Management of preterm labor by tocolysis remains an unmet medical need. Prostaglandins play a major role in regulation of uterine activity and in molecular mechanisms of human labor and parturition. There is some circumstantial evidence that prostaglandin F2α by action through the prostaglandin receptor subtype FP is effective in key events during labor uterine contraction, rupture of membranes and cervical dilation. This role of FP is briefly reviewed. In this study, we tested the hypothesis that an orally active and selective FP antagonist may arrest labor and delay parturition in animal models.</p> <p>Methods</p> <p>We examined the effects of a small molecule selective antagonist of the FP receptor (AS604872) in inhibition of spontaneous uterine contraction in pregnant rat near term. We tested AS604872 for its ability to delay preterm birth in a mouse model in which the anti-progestin agent RU486 triggered parturition.</p> <p>Results</p> <p>By oral or intravenous dosing AS604872 reduced markedly and dose-dependently the spontaneous uterine contractions in late-term pregnant rats at gestational days 19–21. In pregnant mice, AS604872 delayed the preterm birth caused by RU486 administration. The effect was dose-dependent with a significant increase in the mean delivery time of 16 and 33 hours at oral doses of 30 mg/kg and 100 mg/kg, respectively, in the case of labor triggered at gestational day 14. In both models AS604872 appeared more effective than the β-agonist ritodrine.</p> <p>Conclusion</p> <p>The tocolytic activity displayed by a selective FP receptor antagonist supports a key role for the FP receptor in the pathophysiology of premature birth and demonstrates the therapeutic potential of an FP antagonist for the treatment of preterm labor cases in which uterine hyperactivity plays a dominant role.</p

Voltage-gated Na⁺ channel activity increases colon cancertranscriptional activity and invasion via persistent MAPK signaling

© 2015 Macmillan Publishers Limited. All rights reserved. Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes

Frequent loss of heterozygosity and altered expression of the candidate tumor suppressor gene 'FAT' in human astrocytic tumors

Background: We had earlier used the comparison of RAPD (Random Amplification of Polymorphic DNA) DNA fingerprinting profiles of tumor and corresponding normal DNA to identify genetic alterations in primary human glial tumors. This has the advantage that DNA fingerprinting identifies the genetic alterations in a manner not biased for locus. Methods: In this study we used RAPD-PCR to identify novel genomic alterations in the astrocytic tumors of WHO grade II (Low Grade Diffuse Astrocytoma) and WHO Grade IV (Glioblastoma Multiforme). Loss of heterozygosity (LOH) of the altered region was studied by microsatellite and Single Nucleotide Polymorphism (SNP) markers. Expression study of the gene identified at the altered locus was done by semi-quantitative reverse-transcriptase-PCR (RT-PCR). Results: Bands consistently altered in the RAPD profile of tumor DNA in a significant proportion of tumors were identified. One such 500 bp band, that was absent in the RAPD profile of 33% (4/12) of the grade II astrocytic tumors, was selected for further study. Its sequence corresponded with a region of FAT, a putative tumor suppressor gene initially identified in Drosophila. Fifty percent of a set of 40 tumors, both grade II and IV, were shown to have Loss of Heterozygosity (LOH) at this locus by microsatellite (intragenic) and by SNP markers. Semi-quantitative RT-PCR showed low FAT mRNA levels in a major subset of tumors. Conclusion: These results point to a role of the FAT in astrocytic tumorigenesis and demonstrate the use of RAPD analysis in identifying specific alterations in astrocytic tumors

Spin chemistry investigation of peculiarities of photoinduced electron transfer in donor-acceptor linked system

Photoinduced intramolecular electron transfer in linked systems, (R,S)- and (S,S)-naproxen-N-methylpyrrolidine dyads, has been studied by means of spin chemistry methods [magnetic field effect and chemically induced dynamic nuclear polarization (CIDNP)]. The relative yield of the triplet state of the dyads in different magnetic field has been measured, and dependences of the high-field CIDNP of the N-methylpyrrolidine fragment on solvent polarity have been investigated. However, both (S,S)- and (R,S)-enantiomers demonstrate almost identical CIDNP effects for the entire range of polarity. It has been demonstrated that the main peculiarities of photoprocesses in this linked system are connected with the participation of singlet exciplex alongside with photoinduced intramolecular electron transfer in chromophore excited state quenching.This work was supported by the grants 08-03-00372 and 11-03-01104 of the Russian Foundation for Basic Research, and the grant of Priority Programs of the Russian Academy of Sciences, nr. 5.1.5.Magin, I.; Polyakov, N.; Khramtsova, E.; Kruppa, A.; Stepanov, A.; Purtov, P.; Leshina, T.... (2011). Spin chemistry investigation of peculiarities of photoinduced electron transfer in donor-acceptor linked system. Applied Magnetic Resonance. 41(2-4):205-220. https://doi.org/10.1007/s00723-011-0288-3S205220412-4J.S. Park, E. Karnas, K. Ohkubo, P. Chen, K.M. Kadish, S. Fukuzumi, C.W. Bielawski, T.W. Hudnall, V.M. Lynch, J.L. Sessler, Science 329, 1324–1327 (2010)S.Y. Reece, D.G. Nocera, Annu. Rev. Biochem. 78, 673–699 (2009)M.S. Afanasyeva, M.B. Taraban, P.A. Purtov, T.V. Leshina, C.B. Grissom, J. Am. Chem. Soc. 128, 8651–8658 (2006)M.A. Fox, M. Chanon, in Photoinduced Electron Transfer. C: Photoinduced Electron Transfer Reactions: Organic Substrates (Elsevier, New York, 1988), p. 754P.J. Hayball, R.L. Nation, F. Bochner, Chirality 4, 484–487 (1992)N. Suesa, M.F. Fernandez, M. Gutierrez, M.J. Rufat, E. Rotllan, L. Calvo, D. Mauleon, G. Carganico, Chirality 5, 589–595 (1993)A.M. Evans, J. Clin. Pharmacol. 36, 7–15 (1996)Y. Inoue, T. Wada, S. Asaoka, H. Sato, J.-P. Pete, Chem Commun. 4, 251–259 (2000)T. Yorozu, K. Hayashi, M. Irie, J. Am. Chem. Soc. 103, 5480–5548 (1981)N.J. Turro, in Modern Molecular Photochemistry (Benjamin/Cummings, San Francisco, 1978)K.M. Salikhov, Y.N. Molin, R.Z. Sagdeev, A.L. Buchachenko, in Spin Polarization and Magnetic Field Effects in Radical Reactions (Akademiai Kiado, Budapest, 1984), p. 419E.A. Weiss, M.A. Ratner, M.R. Wasielewski, J. Phys. Chem. A 107, 3639–3647 (2003)A.S. Lukas, P.J. Bushard, E.A. Weiss, M.R. Wasielewski, J. Am. Chem. Soc. 125, 3921–3930 (2003)R. Nakagaki, K. Mutai, M. Hiramatsu, H. Tukada, S. Nakakura, Can. J. Chem. 66, 1989–1996 (1988)M.C. Jim′enez, U. Pischel, M.A. Miranda, J. Photochem. Photobiol. C Photochem. Rev. 8, 128–142 (2007)S. Abad, U. Pischel, M.A. Miranda, Photochem. Photobiol. Sci. 4, 69–74 (2005)U. Pischel, S. Abad, L.R. Domingo, F. Bosca, M.A. Miranda, Angew. Chem. Int. Ed. 42, 2531–2534 (2003)G.L. Closs, R.J. Miller, J. Am. Chem. Soc. 101, 1639–1641 (1979)G.L. Closs, R.J. Miller, J. Am. Chem. Soc. 103, 3586–3588 (1981)M. Goez, Chem. Phys. Lett. 188, 451–456 (1992)I.F. Molokov, Y.P. Tsentalovich, A.V. Yurkovskaya, R.Z. Sagdeev, J. Photochem. Photobiol. A 110, 159–165 (1997)U. Pischel, S. Abad, M.A. Miranda, Chem. Commun. 9, 1088–1089 (2003)H. Hayashi, S. Nagakura, Bull. Chem. Soc. Jpn. 57, 322–328 (1984)Y. Sakaguchi, H. Hayashi, S. Nagakura, Bull. Chem. Soc. Jpn. 53, 39–42 (1980)H. Yonemura, H. Nakamura, T. Matsuo, Chem. Phys. Lett. 155, 157–161 (1989)N. Hata, M. Hokawa, Chem. Lett. 10, 507–510 (1981)M. Shiotani, L. Sjoeqvist, A. Lund, S. Lunell, L. Eriksson, M.B. Huang, J. Phys. Chem. 94, 8081–8090 (1990)E. Schaffner, H. Fischer, J. Phys. Chem. 100, 1657–1665 (1996)Y. Mori, Y. Sakaguchi, H. Hayashi, Chem. Phys. Lett. 286, 446–451 (1998)I.M. Magin, A.I. Kruppa, P.A. Purtov, Chem. Phys. 365, 80–84 (2009)K.K. Barnes, Electrochemical Reactions in Nonaqueous Systems (M. Dekker, New York, 1970), p. 560J. Bargon, J. Am. Chem. Soc. 99, 8350–8351 (1977)M. Goez, I. Frisch, J. Phys. Chem. A 106, 8079–8084 (2002)A.K. Chibisov, Russ. Chem. Rev. 50, 615–629 (1981)J. Goodman, K. Peters, J. Am. Chem. Soc. 107, 1441–1442 (1985)H. Cao, Y. Fujiwara, T. Haino, Y. Fukazawa, C.-H. Tung, Y. Tanimoto, Bull. Chem. Soc. Jpn. 69, 2801–2813 (1996)P.A. Purtov, A.B. Doktorov, Chem. Phys. 178, 47–65 (1993)A.I. Kruppa, O.I. Mikhailovskaya, T.V. Leshina, Chem. Phys. Lett. 147, 65–71 (1988)M.E. Michel-Beyerle, R. Haberkorn, W. Bube, E. Steffens, H. Schröder, H.J. Neusser, E.W. Schlag, H. Seidlitz, Chem. Phys. 17, 139–145 (1976)K. Schulten, H. Staerk, A. Weller, H.-J. Werner, B. Nickel, Z. Phys. Chem. 101, 371–390 (1976)K. Gnadig, K.B. Eisenthal, Chem. Phys. Lett. 46, 339–342 (1977)T. Nishimura, N. Nakashima, N. Mataga, Chem. Phys. Lett. 46, 334–338 (1977)M.G. Kuzmin, I.V. Soboleva, E.V. Dolotova, D.N. Dogadkin, High Eng. Chem. 39, 86–96 (2005

Defective germline reprogramming rewires the spermatogonial transcriptome.

Defective germline reprogramming in Piwil4 (Miwi2)- and Dnmt3l-deficient mice results in the failure to reestablish transposon silencing, meiotic arrest and progressive loss of spermatogonia. Here we sought to understand the molecular basis for this spermatogonial dysfunction. Through a combination of imaging, conditional genetics and transcriptome analysis, we demonstrate that germ cell elimination in the respective mutants arises as a result of defective de novo genome methylation during reprogramming rather than because of a function for the respective factors within spermatogonia. In both Miwi2-/- and Dnmt3l-/- spermatogonia, the intracisternal-A particle (IAP) family of endogenous retroviruses is derepressed, but, in contrast to meiotic cells, DNA damage is not observed. Instead, we find that unmethylated IAP promoters rewire the spermatogonial transcriptome by driving expression of neighboring genes. Finally, spermatogonial numbers, proliferation and differentiation are altered in Miwi2-/- and Dnmt3l-/- mice. In summary, defective reprogramming deregulates the spermatogonial transcriptome and may underlie spermatogonial dysfunction

Role of Fas/FasL in regulation of inflammation in vaginal tissue during HSV-2 infection

To assess the role of Fas in lesion development during genital HSV-2 infection, we used a well-established HSV-2 murine model applied to MRL-Faslpr/J (Fas−/−) and C3-Faslgld/J (FasL−/−) C57BL6 mice. In vitro infection of murine keratinocytes and epithelial cells was used to clarify molecular details of HSV-2 infection. Despite upregulation of Fas and FasL, HSV-2-infected keratinocytes and epithelial cells showed a moderate level of apoptosis due to upregulated expression of the anti-apoptotic factors Bcl-2, Akt kinase and NF-κB. Inflammatory lesions within the HSV-2-infected epithelium of C57BL6 mice consisted of infected cells upregulating Fas, FasL and Bcl-2, uninfected cells upregulating Fas and neutrophils expressing both Fas and FasL. Apoptosis was detected in HSV-2-infected cells and to even higher extent in non-infected cells surrounding HSV-2 infection sites. HSV-2 infection of Fas- and FasL-deficient mice led to increased apoptosis and stronger recruitment of neutrophils within the infection sites. We conclude that the Fas pathway participates in regulation of inflammatory response in the vaginal epithelium at the initial stage of HSV-2 infection