Unwinding of primer-templates by archaeal family-B DNA polymerases in response to template-strand uracil

Archaeal family-B DNA polymerases bind tightly to deaminated bases and stall replication on encountering uracil in template strands, four bases ahead of the primer-template junction. Should the polymerase progress further towards the uracil, for example, to position uracil only two bases in front of the junction, 3′–5′ proof-reading exonuclease activity becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and permanent mutation in 50% of the progeny. This publication uses both steady-state and time-resolved 2-aminopurine fluorescence to show pronounced unwinding of primer-templates with Pyrococcus furiosus (Pfu) polymerase–DNA complexes containing uracil at +2; much less strand separation is seen with uracil at +4. DNA unwinding has long been recognized as necessary for proof-reading exonuclease activity. The roles of M247 and Y261, amino acids suggested by structural studies to play a role in primer-template unwinding, have been probed. M247 appears to be unimportant, but 2-aminopurine fluorescence measurements show that Y261 plays a role in primer-template strand separation. Y261 is also required for full exonuclease activity and contributes to the fidelity of the polymerase

Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase

[EN] During DNA replication replicative polymerases move in discrete mechanical steps along the DNA template. To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication. We determine the main kinetic parameters of the nucleotide incorporation cycle and their dependence on external load and nucleotide (dNTP) concentration. The data is inconsistent with power stroke models for translocation, instead supports a loose-coupling mechanism between chemical catalysis and mechanical translocation during DNA replication. According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state. We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (~50 pN).We thank Stephan Grill laboratory (MPI-CBG, Dresden) for help with data collection and E. Galburt, M. Manosas and M. De Vega for critical reading of the manuscript. Spanish Ministry of Economy and Competitiveness [BFU2011-29038 to J.L.C., BFU2013-44202 to J.M.V., BFU2011-23645 to M.S., FIS2010-17440, GR35/10-A920GR35/10-A-911 to F.J.C., MAT2013-49455-EXP to J.R.A.-G. and BFU2012-31825 to B.I.]; Regional Government of Madrid [S2009/MAT 1507 to J.L.C. and CDS2007-0015 to M.S.]; European Molecular Biology Organization [ASTF 276-2012 to J.M.L.]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2012-31825 to B.I.].Morin, J.; Cao, F.; Lázaro, J.; Arias-Gonzalez, JR.; Valpuesta, J.; Carrascosa, J.; Salas, M.... (2015). 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Controlled delivery of ibuprofen from poly(vinyl alcohol)−poly(ethylene glycol) interpenetrating polymeric network hydrogels

Hydrogels composed of poly(vinyl alcohol) (PVA) and poly(ethylene glycol) (PEG) were synthesized using glutaraldehyde as crosslinker and investigated for controlled delivery of the common anti-inflammatory drug, ibuprofen (IBF). To regulate the drug delivery, solid inclusion complexes (ICs) of IBF in β–cyclodextrin (β–CD) were prepared and added to the hydrogels. The ICs were prepared by the microwave irradiation method, which is more environmentally benign. The formation of IC was confirmed by various analytical techniques and the synthesized hydrogels were also characterized. Controlled release of drug was achieved from the hydrogels containing the ICs in comparison to the rapid release from hydrogels containing free IBF. The preliminary kinetic analysis emphasized the crucial role of β–CD in the drug release process that influences the polymer relaxation, thereby leading to prolonged release. The cytotoxicity assay validated the hydrogels as non-toxic in nature and hence can be utilized for controlled delivery of IBF. Keywords: Hydrogels, Poly(vinyl alcohol), Poly(ethylene glycol), Ibuprofen, Controlled delivery system

Cyclodextrin Mediated Controlled Release of Naproxen from pH-Sensitive Chitosan/Poly(Vinyl Alcohol) Hydrogels for Colon Targeted Delivery

pH-Responsive hydrogels are the current need of the hour in controlled drug delivery applications. pH-sensitive interpolymeric hydrogels based on chitosan and polyvinyl alcohol cross-linked with glutaraldehyde were synthesized. Preformed inclusion complex of naproxen with β-cyclodextrin (1:1) was directly loaded into the hydrogel. The hydrogels exhibited maximum swelling at neutral pH. They showed negligible drug release in the simulated gastric fluid and sustained release in the intestinal fluid. The preliminary kinetic analysis revealed that cyclodextrin influences the relaxation rate of the polymer chains, leading to slower release of the drug. It can be proposed that the deleterious effects of naproxen on the epithelium of the gastro-intestinal tract could be minimized by using the cyclodextrin containing hydrogels as drug delivery systems because they provide a controlled release resulting in a reduced concentration of free naproxen. Further, the pH-specific release behavior of these hydrogels can be utilized for colon-targeted drug delivery

Photophysical behaviour of 1-(4-N,N-dimethylaminophenylethynyl)pyrene (DMAPEPy) in homogeneous media

The photophysical behaviour of a new pyrene derivative, 1-(4-N,N-dimethylaminophenylethynyl) pyrene (DMAPEPy), in various solvents has been studied. Due to the presence of an ethynyl link with a cylindrical π cloud between the donor (N,N-dimethyl group) and the acceptor (pyrene), the molecule shows efficient intramolecular charge transfer, with a high extinction coefficient in all the solvents. There is significant solvatochromism in the fluorescence with a large increase in the Stokes' shift of around 125 nm between n-hexane and acetonitrile. The solvent-dependent spectral data show a good correlation with the Kamlet–Taft solvent polarity parameter (π*). The plots of Stokes' shifts with ET (30) are linear for non-protic solvents and for protic solvents but with different slopes. The fluorescence quantum yields are high for non-polar solventsand decrease as the solvent polarity increases. Unlike the parent molecule pyrene, DMAPEPy shows a short lifetime, which is fairly insensitive to oxygen-induced quenching and is dependent on solvent polarity. The molecule shows high steady-state fluorescence anisotropy, which is very sensitive to the viscosity change of the medium

Monocyte-Macrophage Lineage Cell Fusion

International audienceCell fusion (fusogenesis) occurs in natural and pathological conditions in prokaryotes and eukaryotes. Cells of monocyte-macrophage lineage are highly fusogenic. They create syncytial multinucleated giant cells (MGCs) such as osteoclasts (OCs), MGCs associated with the areas of infection/inflammation, and foreign body-induced giant cells (FBGCs). The fusion of monocytes/macrophages with tumor cells may promote cancer metastasis. We describe types and examples of monocyte-macrophage lineage cell fusion and the role of actin-based structures in cell fusion

Giant Multinucleated Cells in Aging and Senescence-An Abridgement

International audienceThis review introduces the subject of senescence, aging, and the formation of senescent multinucleated giant cells. We define senescence and aging and describe how molecular and cellular senescence leads to organismal senescence. We review the latest information on senescent cells’ cellular and molecular phenotypes. We describe molecular and cellular features of aging and senescence and the role of multinucleated giant cells in aging-related conditions and cancer. We explain how multinucleated giant cells form and their role in aging arteries and gonads. We also describe how multinucleated giant cells and the reversibility of senescence initiate cancer and lead to cancer progression and metastasis. We also describe molecules and pathways regulating aging and senescence in model systems and their applicability to clinical therapies in age-related diseases

PSEUDOEPITHELIOMATOUS, KERATOTIC, AND MICACEOUS BALANITIS

A 51-year-old circumcised male presented with hard, thick, keratotic, nail-like covering of the skin of his glans penis of 2 year duration. Histology showed acanthosis, papillomatosis, and elongated rete ridges into the dermis suggestive of pseudoepitheliomatous, keratotic, and micaceous balanitis with features of cellular atypia. Partial penile amputation was done. There was no recurrence after 6 months of follow up

Demethylase-independent roles of LSD1 in regulating enhancers and cell fate transition

Abstract The major enhancer regulator lysine-specific histone demethylase 1A (LSD1) is required for mammalian embryogenesis and is implicated in human congenital diseases and multiple types of cancer; however, the underlying mechanisms remain enigmatic. Here, we dissect the role of LSD1 and its demethylase activity in gene regulation and cell fate transition. Surprisingly, the catalytic inactivation of LSD1 has a mild impact on gene expression and cellular differentiation whereas the loss of LSD1 protein de-represses enhancers globally and impairs cell fate transition. LSD1 deletion increases H3K27ac levels and P300 occupancy at LSD1-targeted enhancers. The gain of H3K27ac catalyzed by P300/CBP, not the loss of CoREST complex components from chromatin, contributes to the transcription de-repression of LSD1 targets and differentiation defects caused by LSD1 loss. Together, our study demonstrates a demethylase-independent role of LSD1 in regulating enhancers and cell fate transition, providing insight into treating diseases driven by LSD1 mutations and misregulation