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941.
Maynard-Smith MD McKelvie JC Wood RJ Harmer JE Ranasinghe RT Williams CL Coomber DM Stares AF Roach PL 《Analytical biochemistry》2011,(2):204-212
N-6 methylation of adenine destabilises duplex DNA and this can increase the proportion of DNA that dissociates into single strands. We have investigated utilising this property to measure the DNA adenine methyltransferase-catalyzed conversion of hemimethylated to fully methylated DNA through a simple, direct, fluorescence-based assay. The effects of methylation on the kinetics and thermodynamics of hybridisation were measured by comparing a fully methylated oligonucleotide product and a hemimethylated oligonucleotide substrate using a 13-bp duplex labeled on adjacent strands with a fluorophore (fluorescein) and quencher (dabcyl). Enzymatic methylation of the hemimethylated GATC site resulted in destabilisation of the duplex, increasing the proportion of dissociated DNA, and producing an observable increase in fluorescence. The assay provides a direct measurement of methylation rate in real time and is highly reproducible, with a coefficient of variance over 48 independent measurements of 3.6%. DNA methylation rates can be measured as low as 3.55 ± 1.84 fmol s−1 in a 96-well plate format, and the assay has been used to kinetically characterise the Pyrococcus horikoshii DNA adenine methyltransferase. 相似文献
942.
943.
Biswas A Pasquel D Tyagi RK Mani S 《Biochemical and biophysical research communications》2011,(3):2008-376
Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependent functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation. 相似文献
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946.
Y. K. Stella Man Danielle DiCara Nicole Chan Sandrine Vessillier Stephen J. Mather Michelle L. Rowe Mark J. Howard John F. Marshall Ahuva Nissim 《PloS one》2013,8(8)
We have developed a structurally-guided scaffold phage display strategy for identification of ligand mimetic bio-therapeutics. As a proof of concept we used the ligand of integrin αvβ6, a tumour cell surface receptor and a major new target for imaging and therapy of many types of solid cancer. NMR structure analysis showed that RGD-helix structures are optimal for αvβ6 ligand-interaction, so we designed novel algorithms to generate human single chain fragment variable (scFv) libraries with synthetic VH-CDR3 encoding RGD-helix hairpins with helices of differing pitch, length and amino acid composition. Study of the lead scFv clones D25scFv and D34scFv and their corresponding VH-CDR3 derived peptides, D25p and D34p, demonstrated: specific binding to recombinant and cellular αvβ6; inhibition of αvβ6-dependent cell and ligand adhesion, αvβ6-dependent cell internalisation; and selective retention by αvβ6-expressing, but not αvβ6-negative, human xenografts. NMR analysis established that both the D25p and D34p retained RGD-helix structures confirming the success of the algorithm. In conclusion, scFv libraries can be engineered based on ligand structural motifs to increase the likelihood of developing powerful bio-therapeutics. 相似文献
947.
Dirce Maria Carraro Maria Aparecida Azevedo Koike Folgueira Bianca Cristina Garcia Lisboa Eloisa Helena Ribeiro Olivieri Ana Cristina Vitorino Krepischi Alex Fiorini de Carvalho Louise Danielle de Carvalho Mota Renato David Puga Maria do Socorro Maciel Rodrigo Augusto Depieri Michelli Eduardo Carneiro de Lyra Stana Helena Giorgi Grosso Fernando Augusto Soares Maria Isabel Alves de Souza Waddington Achatz Helena Brentani Carlos Alberto Moreira-Filho Maria Mitzi Brentani 《PloS one》2013,8(3)
948.
Patricia L. Schirmer Cynthia A. Lucero-Obusan Stephen R. Benoit Luis M. Santiago Danielle Stanek Achintya Dey Mirsonia Martinez Gina Oda Mark Holodniy 《PLoS neglected tropical diseases》2013,7(3)
Background
Although dengue is endemic in Puerto Rico (PR), 2007 and 2010 were recognized as epidemic years. In the continental United States (US), outside of the Texas-Mexico border, there had not been a dengue outbreak since 1946 until dengue re-emerged in Key West, Florida (FL), in 2009–2010. The objective of this study was to use electronic and manual surveillance systems to identify dengue cases in Veterans Affairs (VA) healthcare facilities and then to clinically compare dengue cases in Veterans presenting for care in PR and in FL.Methodology
Outpatient encounters from 1/2007–12/2010 and inpatient admissions (only available from 10/2009–12/2010) with dengue diagnostic codes at all VA facilities were identified using VA''s Electronic Surveillance System for Early Notification of Community-based Epidemics (ESSENCE). Additional case sources included VA data from Centers for Disease Control and Prevention BioSense and VA infection preventionists. Case reviews were performed. Categorical data was compared using Mantel-Haenszel or Fisher Exact tests and continuous variables using t-tests. Dengue case residence was mapped.Findings
Two hundred eighty-eight and 21 PR and FL dengue cases respectively were identified. Of 21 FL cases, 12 were exposed in Key West and 9 were imported. During epidemic years, FL cases had significantly increased dengue testing and intensive care admissions, but lower hospitalization rates and headache or eye pain symptoms compared to PR cases. There were no significant differences in clinical symptoms, laboratory abnormalities or outcomes between epidemic and non-epidemic year cases in FL and PR. Confirmed/probable cases were significantly more likely to be hospitalized and have thrombocytopenia or leukopenia compared to suspected cases.Conclusions
Dengue re-introduction in the continental US warrants increased dengue surveillance and education in VA. Throughout VA, under-testing of suspected cases highlights the need to emphasize use of diagnostic testing to better understand the magnitude of dengue among Veterans. 相似文献949.
An Antimicrobial Peptidomimetic Induces Mucorales Cell Death through Mitochondria-Mediated Apoptosis
E. Magda Barbu Fazal Shirazi Danielle M. McGrath Nathaniel Albert Richard L. Sidman Renata Pasqualini Wadih Arap Dimitrios P. Kontoyiannis 《PloS one》2013,8(10)
The incidence of mucormycosis has dramatically increased in immunocompromised patients. Moreover, the array of cellular targets whose inhibition results in fungal cell death is rather limited. Mitochondria have been mechanistically identified as central regulators of detoxification and virulence in fungi. Our group has previously designed and developed a proteolytically-resistant peptidomimetic motif D(KLAKLAK)2 with pleiotropic action ranging from targeted (i.e., ligand-directed) activity against cancer and obesity to non-targeted activity against antibiotic resistant gram-negative rods. Here we evaluated whether this non-targeted peptidomimetic motif is active against Mucorales. We show that D(KLAKLAK)2 has marked fungicidal action, inhibits germination, and reduces hyphal viability. We have also observed cellular changes characteristic of apoptosis in D(KLAKLAK)2-treated Mucorales cells. Moreover, the fungicidal activity was directly correlated with vacuolar injury, mitochondrial swelling and mitochondrial membrane depolarization, intracellular reactive oxygen species accumulation (ROS), and increased caspase-like enzymatic activity. Finally, these apoptotic features were prevented by the addition of the ROS scavenger N-acetyl-cysteine indicating mechanistic pathway specificity. Together, these findings indicate that D(KLAKLAK)2 makes Mucorales exquisitely susceptible via mitochondrial injury-induced apoptosis. This prototype may serve as a candidate drug for the development of translational applications against mucormycosis and perhaps other fungal infections. 相似文献
950.
Lucette A. Cysique Kirsten Moffat Danielle M. Moore Tammy A. Lane Nicholas W. S. Davies Andrew Carr Bruce J. Brew Caroline Rae 《PloS one》2013,8(4)