59 research outputs found
Differential kinetics of endothelial cell activation biomarkers E-selectin and endocan during nonlethal endotoxemia in 129Sv mice: a role for PMN-derived serine proteases in the transient decrease of circulating endocan levels
Endocan (endothelial cell-specific molecule-1) as a pertinent biomarker of endothelial dysfunction in sepsis
Tuning the translational freedom of DNA for high speed AFM
Direct observation is arguably the preferred way to investigate the interactions between two molecular complexes. With the development of high speed atomic force microscopy it is becoming possible to observe directly DNA protein interactions with relevant spatial and temporal resolutions. These interactions are of central importance to biology, bio-nanotechnology but also functional biologically inspired materials. Critically, sample preparation plays a central role in all microscopy studies and minimal perturbation of the sample is desired. Here, we demonstrate the ability to tune the interactions of DNA molecules with the surface such that an association strong enough to enable high resolution AFM imaging while providing sufficient translational freedom to allow the relevant protein DNA interactions to take place, can be maintained. Furthermore, we describe a quantitative method for measuring the DNA mobility, which also allows the dissection of the different contributions to the overall movement of the DNA molecules. We find that for weak surface association, a significant contribution to the movement arises from the interaction of the AFM tip with the DNA. In combination, these methods enable the tuning of the surface translational freedom of DNA molecules to allow the direct study of a wide range of nucleo-protein interactions by high speed atomic force microscopy
Polyamine Sharing between Tubulin Dimers Favours Microtubule Nucleation and Elongation via Facilitated Diffusion
We suggest for the first time that the action of multivalent cations on
microtubule dynamics can result from facilitated diffusion of GTP-tubulin to the
microtubule ends. Facilitated diffusion can promote microtubule assembly,
because, upon encountering a growing nucleus or the microtubule wall, random
GTP-tubulin sliding on their surfaces will increase the probability of
association to the target sites (nucleation sites or MT ends).
This is an original explanation for understanding the apparent discrepancy
between the high rate of microtubule elongation and the low rate of tubulin
association at the microtubule ends in the viscous cytoplasm. The mechanism of
facilitated diffusion requires an attraction force between two tubulins, which
can result from the sharing of multivalent counterions. Natural polyamines
(putrescine, spermidine, and spermine) are present in all
living cells and are potent agents to trigger tubulin self-attraction. By using
an analytical model, we analyze the implication of facilitated diffusion
mediated by polyamines on nucleation and elongation of microtubules. In
vitro experiments using pure tubulin indicate that the promotion of
microtubule assembly by polyamines is typical of facilitated diffusion. The
results presented here show that polyamines can be of particular importance for
the regulation of the microtubule network in vivo and provide
the basis for further investigations into the effects of facilitated diffusion
on cytoskeleton dynamics
Mechanics of the IL2RA Gene Activation Revealed by Modeling and Atomic Force Microscopy
Transcription implies recruitment of RNA polymerase II and transcription factors (TFs) by DNA melting near transcription start site (TSS). Combining atomic force microscopy and computer modeling, we investigate the structural and dynamical properties of the IL2RA promoter and identify an intrinsically negative supercoil in the PRRII region (containing Elf-1 and HMGA1 binding sites), located upstream of a curved DNA region encompassing TSS. Conformational changes, evidenced by time-lapse studies, result in the progressive positioning of curvature apex towards the TSS, likely facilitating local DNA melting. In vitro assays confirm specific binding of the General Transcription Factors (GTFs) TBP and TFIIB over TATA-TSS position, where an inhibitory nucleosome prevented preinitiation complex (PIC) formation and uncontrolled DNA melting. These findings represent a substantial advance showing, first, that the structural properties of the IL2RA promoter are encoded in the DNA sequence and second, that during the initiation process DNA conformation is dynamic and not static
Analysis of In-Vivo LacR-Mediated Gene Repression Based on the Mechanics of DNA Looping
Interactions of E. coli lac repressor (LacR) with a pair of operator sites on the same DNA molecule can lead to the formation of looped nucleoprotein complexes both in vitro and in vivo. As a major paradigm for loop-mediated gene regulation, parameters such as operator affinity and spacing, repressor concentration, and DNA bending induced by specific or non-specific DNA-binding proteins (e.g., HU), have been examined extensively. However, a complete and rigorous model that integrates all of these aspects in a systematic and quantitative treatment of experimental data has not been available. Applying our recent statistical-mechanical theory for DNA looping, we calculated repression as a function of operator spacing (58–156 bp) from first principles and obtained excellent agreement with independent sets of in-vivo data. The results suggest that a linear extended, as opposed to a closed v-shaped, LacR conformation is the dominant form of the tetramer in vivo. Moreover, loop-mediated repression in wild-type E. coli strains is facilitated by decreased DNA rigidity and high levels of flexibility in the LacR tetramer. In contrast, repression data for strains lacking HU gave a near-normal value of the DNA persistence length. These findings underscore the importance of both protein conformation and elasticity in the formation of small DNA loops widely observed in vivo, and demonstrate the utility of quantitatively analyzing gene regulation based on the mechanics of nucleoprotein complexes
Structure of Metaphase Chromosomes: A Role for Effects of Macromolecular Crowding
In metaphase chromosomes, chromatin is compacted to a concentration of several hundred mg/ml by mechanisms which remain elusive. Effects mediated by the ionic environment are considered most frequently because mono- and di-valent cations cause polynucleosome chains to form compact ∼30-nm diameter fibres in vitro, but this conformation is not detected in chromosomes in situ. A further unconsidered factor is predicted to influence the compaction of chromosomes, namely the forces which arise from crowding by macromolecules in the surrounding cytoplasm whose measured concentration is 100–200 mg/ml. To mimic these conditions, chromosomes were released from mitotic CHO cells in solutions containing an inert volume-occupying macromolecule (8 kDa polyethylene glycol, 10.5 kDa dextran, or 70 kDa Ficoll) in 100 µM K-Hepes buffer, with contaminating cations at only low micromolar concentrations. Optical and electron microscopy showed that these chromosomes conserved their characteristic structure and compaction, and their volume varied inversely with the concentration of a crowding macromolecule. They showed a canonical nucleosomal structure and contained the characteristic proteins topoisomerase IIα and the condensin subunit SMC2. These observations, together with evidence that the cytoplasm is crowded in vivo, suggest that macromolecular crowding effects should be considered a significant and perhaps major factor in compacting chromosomes. This model may explain why ∼30-nm fibres characteristic of cation-mediated compaction are not seen in chromosomes in situ. Considering that crowding by cytoplasmic macromolecules maintains the compaction of bacterial chromosomes and has been proposed to form the liquid crystalline chromosomes of dinoflagellates, a crowded environment may be an essential characteristic of all genomes
Os Usos de Simuladores Durante a Formação Profissional para a Condução de Transportes Rodoviários
DNA-Linker-Induced Surface Assembly of Ultra Dense Parallel Single Walled Carbon Nanotube Arrays
Direct Atomic Force Microscopy Observation of DNA Tile Crystal Growth at the Single-Molecule Level
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