No abstract

Factors affecting the functionalization of mica with aminosilanes, in particular, aminopropyltriethoxysilane (APTES-mica), formed from the vapor phase have been systematically studied. The relative humidity (RH) during vapor deposition has been varied, and postdeposition treatment through baking has been used, as well as the comparison of mono and trifunctionality, to investigate how optimal surfaces for AFM imaging of DNA are formed. It is found that the stability of the APTES layers is a consequence of lateral polymerization and not covalent attachment to the mica substrate. At low RH (<25%), DNA adopts an open, well-resolved conformation, whereas at >25% RH, DNA surface-induced condensation occurs. Contact mode AFM scratching experiments show that two main structures of the silane layer exist at different humidity: a monolayer exists at RH < 25%, and a bilayer structure exists at RH > 25%. Finally, structural changes that these two layer types undergo after baking at 150 degrees C were investigated by AFM and X-ray photoelectron spectroscopy (XPS), and these now prevented DNA from binding to the APTES-mica, except in the presence of Mg(II) ions.

Atomic force microscopy (AFM) has been used to image, at single molecule resolution, transcription events by Escherichia coli RNA polymerase (RNAP) on a linear DNA template with two convergently aligned λpr promoters. For the first time experimentally, the outcome of collision events during convergent transcription by two identical RNAP has been studied. Measurement of the positions of the RNAP on the DNA, allows distinction of open promoter complexes (OPCs) and elongating complexes (EC) and collided complexes (CC). This discontinuous time-course enables subsequent analysis of collision events where both RNAP remain bound on the DNA. After collision, the elongating RNAP has caused the other (usually stalled) RNAP to back-track along the template. The final positions of the two RNAP indicate that these are collisions between an EC and a stalled EC (SEC) or OPC (previously referred to as sitting-ducks). Interestingly, the distances between the two RNAP show that they are not always at closest approach after ‘collision’ has caused their arrest.

Objective. To investigate the effect of isolated hydrostatic pressure on proteoglycan metabolism in chondrocytes.Methods. Bovine articular chondrocytes cultured in agarose gels were subjected to 5 MPa hydrostatic pressure for 4 hours in either a static or a pulsatile (1 Hz) mode, and changes in glycosaminoglycan (GAG) synthesis, hydrodynamic size, and aggregation properties of proteoglycans and aggrecan messenger RNA (mRNA) levels were determined.Results. The application of 5 MPa static pressure caused a significant increase in GAG synthesis of 11% (P < 0.05). Column chromatography showed that this increase in GAG synthesis was associated with large proteoglycans. In addition, semiquantitative reverse transcriptase-polymerase chain reaction showed a 4-fold increase in levels of aggrecan mRNA (P < 0.01).Conclusion. Hydrostatic pressure in isolation, which does not cause cell deformation, can affect proteoglycan metabolism in chondrocytes cultured in agarose gels, indicating an important role of hydrostatic pressure in the regulation of extracellular matrix turnover in articular cartilage.It is generally recognized that mechanical loading is essential in maintaining homeostasis of articular cartilage. However, the physical response of the cartilage matrix to mechanical load is complex and involves many factors, including tissue and cell deformation, change in hydrostatic pressure, and fluid flow (1). This fluid flow not only causes changes in the concentration of extracellular components, but also carries positive counterions and induces streaming potentials and currents (2).Recent reports have described the effects of mechanical compression or hydrostatic pressure applied either directly to cartilaginous tissue or to isolated chondrocytes in culture systems (3-13). Some of these studies have shown that hydrostatic pressure is an important element of mechanical load in the regulation of proteoglycan metabolism in chondrocytes (5,7,9,10,13). It is considered that the interaction of cells with extracellular matrix should be eliminated by isolating chondrocytes from the matrix surrounding them, in order to investigate the effect of a single factor (e.g., hydrostatic pressure) among the various events that occur during the application of mechanical load. However, in all the previous studies regarding the effect of hydrostatic pressure on the metabolism of isolated chondrocytes (not cartilage specimens), pressures were applied to chondrocytes in monolayer culture systems, which could cause dedifferentiation of cells into fibroblastic phenotype (14) and make interpretation of data somewhat difficult.The aim of the current study was to investigate the effect of hydrostatic pressure alone on chondrocyte metabolism in an attempt to dissect the effects of the individual processes affecting physiologic responses of chondrocytes to changes in the mechanical environment. A novel experimental system was developed to apply hydrostatic pressure to chondrocytes cultured in 3-dimensional agarose gels. It thus maintained phenotypic ...

The study of plaque biofilms in the oral cavity is difficult as plaque removal inevitably disrupts biofilm integrity precluding kinetic studies involving the penetration of components and metabolism of substrates in situ. A method is described here in which plaque is formed in vivo under normal (or experimental) conditions using a collection device which can be removed from the mouth after a specified time without physical disturbance to the plaque biofilm, permitting site-specific analysis or exposure of the undisturbed plaque to experimental conitions in vitro. Microbiological analysis revealed plaque flora which was similar to that reported from many natural sources. Analytical data can be related to plaque volume rather than weight. Using this device, plaque fluoride concentrations have been shown to vary with plaque depth and in vitro short-term exposure to radiolabelled components may be carried out, permitting important conclusions to be drawn regarding the site-specific composition and dynamics of dental plaque.