Showing papers by "Emilios K. Dimitriadis published in 2012"

Galactose repressor mediated intersegmental chromosomal connections in Escherichia coli

Abstract: By microscopic analysis of fluorescent-labeled GalR, a regulon-specific transcription factor in Escherichia coli, we observed that GalR is present in the cell as aggregates (one to three fluorescent foci per cell) in nongrowing cells. To investigate whether these foci represent GalR-mediated association of some of the GalR specific DNA binding sites (gal operators), we used the chromosome conformation capture (3C) method in vivo. Our 3C data demonstrate that, in stationary phase cells, many of the operators distributed around the chromosome are interacted. By the use of atomic force microscopy, we showed that the observed remote chromosomal interconnections occur by direct interactions between DNA-bound GalR not involving any other factors. Mini plasmid DNA circles with three or five operators positioned at defined loci showed GalR-dependent loops of expected sizes of the intervening DNA segments. Our findings provide unique evidence that a transcription factor participates in organizing the chromosome in a three-dimensional structure. We believe that these chromosomal connections increase local concentration of GalR for coordinating the regulation of widely separated target genes, and organize the chromosome structure in space, thereby likely contributing to chromosome compaction.

Measurement of plasma volume using fluorescent silica-based nanoparticles

Abstract: Plasma volume (PV) is an important determinant of cardiovascular function and organ perfusion, and it is the target of infusion and diuretic therapies in daily clinical practice. Despite its fundamental importance PV is not commonly measured because available methods of tracer dilution are reliant on dye substances that suffer from numerous drawbacks including binding plasma proteins, spectral changes, and clearance kinetics that complicate analysis and interpretation. To address these issues, we have tested the utility of fluorescent nanoparticles comprised of a dye-rich silica core and polyethylene glycol-coated shell. Photophysical and visual analysis showed discrete size-gradated nanoparticle populations could be synthesized within a distribution tolerance of ±4 nm, which were optically unaffected in the presence of plasma/albumin. In normal mice, the cutoff for renal filtration of nanoparticles from blood into urine was ≤11 nm. A linear relationship between body weight and PV was readily determined in mice administered far red fluorescent nanoparticles sized either 20 or 30 nm. PV measurements using nanoparticles were correlated to values obtained with Evans blue dye. Induced expansion or contraction of PV was demonstrated with albumin or furosemide administration, respectively, in mice. Longitudinal experiments >30 min required matched untreated control mice to correct for nanoparticle loss (≈30%) putatively to the reticuloendothelial/phagocyte system. Collectively, the findings support a nanotechnology-based solution to methodological problems in measure of PV, notably in clinical settings where information on hemodynamic changes may improve treatment of injury and disease.

The linker of the interferon response factor 3 transcription factor is not unfolded.

Abstract: Interferon response factor 3 (IRF-3) is a transcription factor that plays an essential role in controlling the synthesis of interferon-β (IFN-β) and is a protein consisting of two well-defined domains, the N-terminal DNA-binding and the C-terminal dimerization domains, connected by a 75-residue linker, supposedly unfolded. However, it was not clear whether in intact IRF-3 this linker segment of the chain, which carries the nuclear export signal and includes a region of high helical propensity, remains unfolded. This has been investigated using nuclear magnetic resonance by ligating the 15N-labeled linker to the unlabeled N-terminal and C-terminal domains. It was found that, while the linker alone is indeed in a completely unfolded state, when ligated to the C-terminal domain it shows some ordering, and this ordering becomes much more pronounced when the linker is also ligated to the N-terminal domain. Thus, in intact IRF-3, the linker represents a folded structural domain; i.e., IRF-3 is a three-domain gl...

Atomic Force Microscopy of Chromatin

Abstract: Atomic force microscopy or scanning tunneling microscopy (AFM/STM) is a powerful single molecule tool for the visualization of biological materials at sub-nanometer resolution. AFM is versatile because it can directly measure physical properties due to its sensitivity at picoNewton force scales, thus enabling dissection of molecular forces. STM/AFM has been considered revolutionary since its discovery 32 years ago, and a mere 7 years after its invention, Binnig and Rohrer were awarded the Nobel Prize in Physics, along with Ruska, inventor of the first electron microscope. In subsequent years, AFM has evolved fast, with the range of applications for STM/AFM expanding to encompass all physical sciences. Despite it’s popularity in physics, engineering, material and chemistry sciences, it has remained a less prominent tool for biologists, who are much more familiar with techniques like electron microscopy (EM), X-ray crystallography or nuclear magnetic resonance (NMR). The relatively low cost of the microscope, the ease of sample preparation, and the lack of any requirement for staining, freezing, excessive denaturation or preservation, should make AFM an ideal tool for laboratories interested in imaging and manipulating biological samples down to macromolecules at the nanoscale. In this chapter, we will discuss how AFM can be an effective high-resolution technique for structural and molecular interaction studies in biology.

Depth dependent osmotic and swelling properties of cartilage

Abstract: Articular cartilage is a low-friction, load-bearing tissue located at joint surfaces. It experiences static and dynamic forces including shear, compression and tension. We investigate the relationship between structure and function by measuring the osmotic and mechanical properties in cartilage layers as a function of the distance from the articular surface. Atomic force microscopy is used to probe the mechanical properties at high spatial resolution. The mechanical measurements are complemented by osmotic swelling pressure observations made on the same samples using a novel tissue osmometer. The results show that the osmotic modulus significantly depends on the distance from the articular surface. Its value is highest in the deep zone and lowest in the middle zone.

Probing DNA assembly into nanoparticles with short DNA

Abstract: DNA is an anionic polyelectrolyte, which occupies a large volume in salt free solution due to the coulomb repulsion between the charged groups. In the presence of high valence cations, DNA condenses into nanoparticles. DNA nanoparticles have generated a lot of interest as a preferred vehicle for delivering therapeutic DNA in gene therapy. The efficiency of gene delivery is determined by stability and compactness of the particles. However not much is known about the organization of DNA within the particles. The large polymer cations condense DNA rapidly, with no distinct intermediate stages that give insight into the arrangement of DNA within the nanoparticle. In our work, we form nanoparticles with short DNA strands to slow down the condensation process. The polymer cation is polyethyleneimine with grafted sugar moieties. Distinct intermediate stages are observed with Atomic Force Microscopy. The assembly occurs via the formation of fiber condensates, which appear to be the unit of DNA condensation. Nanoparticles form by compaction of interweaving networks of fiber condensates.