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1.
J Biol Chem ; 295(27): 9012-9020, 2020 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-32385112

RESUMEN

Eukaryotic DNA polymerase ß (Pol ß) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol ß has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as "fingers closing." Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol ß. First, using a doubly labeled DNA construct, we show that Pol ß bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol ß and donor-labeled DNA, we visualized dynamic fingers closing in single Pol ß-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol ß, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol ß reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.


Asunto(s)
ADN Polimerasa beta/metabolismo , ADN/metabolismo , Cristalografía por Rayos X/métodos , ADN Polimerasa I/química , ADN Polimerasa beta/fisiología , Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Humanos , Cinética , Modelos Moleculares , Conformación de Ácido Nucleico , Nucleótidos/metabolismo , Conformación Proteica , Especificidad por Sustrato/fisiología
2.
Phytother Res ; 27(2): 264-71, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22566008

RESUMEN

An extract was prepared from Egyptian stabilized rice bran and standardized to contain 2% γ-oryzanol in addition to its content of other bioactives, notably tocotrienol and policosanol. The standardized extract was found to have a concentration-dependent effect on insulin release in vitro, which, however, is not mediated by γ-tocotrienol in rice bran (detected by HPLC) as could have been expected. Policosanol and γ-oryzanol have insulinotropic effects. The in vitro data of rice bran directly translate into in vivo data of rats by using a glucose tolerance test (increase in plasma insulin). Tocotrienols are well known for their apoptotic effect on tumor cells; nevertheless, an attempt was made to study glucose uptake in HEP-G2 cells, which needs to induce an insulin-resistant state by TNF-α. The Egyptian rice bran extract has an antidiabetic effect. γ-Oryzanol, which is a possible precursor of the insulinotropic compound ferulic acid, is a candidate for this effect. Therefore, it is reasonable to assume that the prevalence of diabetes or at least a prediabetic (type 2) situation can be ameliorated by the investigated rice bran extract. The potential usefulness of the extract as a nutraceutical is currently undergoing more thorough investigations.


Asunto(s)
Hipoglucemiantes/farmacología , Oryza/química , Fenilpropionatos/farmacología , Extractos Vegetales/farmacología , Animales , Glucemia/efectos de los fármacos , Cromanos/aislamiento & purificación , Cromanos/farmacología , Ácidos Cumáricos/aislamiento & purificación , Ácidos Cumáricos/farmacología , Suplementos Dietéticos , Alcoholes Grasos/aislamiento & purificación , Alcoholes Grasos/farmacología , Femenino , Prueba de Tolerancia a la Glucosa , Células Hep G2 , Humanos , Hipoglucemiantes/aislamiento & purificación , Insulina/sangre , Masculino , Fenilpropionatos/aislamiento & purificación , Ratas , Tocotrienoles/aislamiento & purificación , Tocotrienoles/farmacología , Factor de Necrosis Tumoral alfa , Vitamina E/análogos & derivados , Vitamina E/aislamiento & purificación , Vitamina E/farmacología
3.
ACS Nano ; 16(9): 14611-14621, 2022 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-36107137

RESUMEN

Whereas the formation and overall stability of hierarchically organized self-assembled supramolecular structures have been extensively investigated, the mechanistic aspects of subcomponent dynamics are often poorly understood or controlled. Here we show that the dynamics of polyamidoamine (PAMAM) dendrimer based micelles can be manipulated by changes in dendrimer generation, pH, and stoichiometry, as proven by NMR and FRET. For this, dendrimers were functionalized with either fluorescein (donor) or rhodamine (acceptor) and encapsulated into separate micelles. Upon mixing, exchange of dendrimers is revealed by an increase in FRET. While dendrimicelles based on dendrimer generations 4 and 5 show a clear increase in FRET in time, revealing the dynamic exchange of dendrimers between micellar cores, generation 6 based micelles appear to be kinetically trapped systems. Interestingly, generation 6 based dendrimicelles prepared at a pH of 7.8 rather than 7.0 do show exchange dynamics, which can be attributed to about 25% less charge of the dendrimer, corresponding to the charge of a virtual generation 5.5 dendrimer at neutral pH. Changing the pH of dendrimicelle solutions prepared at a pH of 7.8 to 7.0 shows the activated release of dendrimers. High-resolution NMR spectra of the micellar core are obtained from a 1.2 GHz spectrometer with sub-micromolar sensitivity, with DOSY discriminating released dendrimers from dendrimers still present in the micellar core. This study shows that dendrimer generation, charge density, and stoichiometry are important mechanistic factors for controlling the dynamics of complex coacervate core micelles. This knowledge can be used to tune micelles between kinetically trapped and dynamic systems, with tuning of exchange and/or release speeds, to be tailored for applications in, e.g., material science, sensors, or drug delivery.


Asunto(s)
Dendrímeros , Micelas , Dendrímeros/química , Fluoresceínas , Concentración de Iones de Hidrógeno , Rodaminas
4.
ACS Nano ; 15(1): 1666-1674, 2021 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-33411511

RESUMEN

A versatile method is presented to form dendrimer superstructures by exploiting coacervate-core micelles as a template to confine and organize the hyperbranched macromolecules. First, complex coacervate-core micelles are formed from negative-neutral block copolymers and positively charged polyamidoamine dendrimers. The dendrimers inside the micellar core are then covalently cross-linked with each other upon addition of glutaraldehyde. After removal of the block copolymer from the assembly by increasing the salt concentration, consecutively, the formed Schiff bases cross-linking the dendrimers are reduced to amines, followed by a final dialysis step. This leads to well-defined covalently cross-linked nanostructures, coined dendroids, with a size of around 30 nm in diameter and a molecular weight of approximately 2.5 MDa. By incorporating dendrimer-encapsulated gold nanoparticles (AuDENs) into the micelle template strategy, the aggregation number of dendrimers inside the dendroids is determined by counting the nanoparticles in TEM micrographs. Furthermore, TEM performed at different tilt angles and AFM analysis corroborate formation of stable, covalently linked three-dimensional structures. Reconstruction of the TEM tilt series results in a tomogram further illustrating the 3D distribution of the gold nanoparticles, and hence the individual dendrimers, in the nanostructure. These dendroids appear to have a hard, poorly compressible core and a relatively soft outside. The versatility of the hierarchical building up of the supermolecules is illustrated by the controlled and synchronous incorporation of empty dendrimers and AuDENs into a single hybrid dendroid structure. The presented strategy allows for the preparation of a variety of classes of supermolecules, depending on the type of micellar-core macromolecule, e.g., dendrimer, cross-linker, and nanoparticles, used. Considering the broad interest in dendrimers as well as micelles in a plethora of research areas, e.g., (targeted) drug delivery, biomedical imaging, theragnostics, and catalysis, there is a great potential for dendroids and related classes of covalently linked macromolecules, viz., supermolecules.

5.
Nanoscale ; 13(36): 15422-15430, 2021 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-34505610

RESUMEN

Hierarchically built-up multicompartment nanoaggregate systems are of interest for, e.g., novel materials and medicine. Here we present a versatile strategy to generate and unambiguously characterize complex coacervate-core micelles by exploiting four different dendrimeric subcomponents as core-units. The resulting mesoscale structures have a hydrodynamic diameter of 50 nm and a core size of 33 nm, and host about thirty 6th generation polyamidoamine (PAMAM) dendrimers. We have used FRET (efficiency of ∼0.2) between fluorescein and rhodamine moieties immobilized on separate PAMAM dendrimers (G6-F and G6-R, respectively) to prove synchronous encapsulation in the micelle core. Tuning the proximity of the FRET pair molecules either by varying the G6-F : G6-R ratio, or by co-assembling non-functionalized dendrimer (G6-E) in the core, reveals the optimal FRET efficiency to occur at a minimum of 70% loading with G6-F and G6-R. Additional co-encapsulation of 6th generation gold dendrimer-encapsulated nanoparticles (G6-Au) in the micelle core shows a dramatic reduction of the FRET efficiency, which can be restored by chemical etching of the gold nanoparticles from within the micellar core with thiols, leaving the micelle itself intact. This study reveals the controlled co-assembly of up to four different types of subcomponents in one single micellar core and concomitantly shows the wide variety of structures that can be made with a well-defined basic set of subcomponents. It is straightforward to design related strategies, to incorporate inside one micellar core, e.g., even more than 4 different dendrimers, or other classes of (macro)molecules, with different functional groups, other FRET pairs or different encapsulated metal nanoparticles.

6.
Bioorg Med Chem ; 16(14): 6747-51, 2008 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-18565754

RESUMEN

A new series of flavonyl-2,4-thiazolidinediones (Va-c, VIa-c) was prepared by Knoevenagel reaction. The synthesized compounds were tested for their ability to inhibit rat kidney aldose reductase (AR) and for their insulinotropic activities in INS-1 cells. Compound Vb was able to increase insulin release in the presence of 5.6mmol/l glucose. Compounds VIa-c displayed moderate to high AR inhibitory activity levels. Particularly, compound VIa showed the highest AR inhibitory activity (86.57%).


Asunto(s)
Hipoglucemiantes/química , Tiazolidinedionas/química , Tiazolidinedionas/farmacología , Aldehído Reductasa/antagonistas & inhibidores , Animales , Línea Celular , Glucosa/farmacología , Humanos , Hipoglucemiantes/farmacología , Insulina/metabolismo , Secreción de Insulina , Riñón/enzimología , Masculino , Ratones , Ratas
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