Effect of heparin and heparan sulphate on open promoter complex formation for a simple tandem gene model using ex situ atomic force microscopy.

The influence of heparin and heparan sulphate (HepS) on the appearance and analysis of open promoter complex (RPo) formation by E. coli RNA polymerase (RNAP) holoenzyme (σ70RNAP) on linear DNA using ex situ imaging by atomic force microscopy (AFM) has been investigated. Introducing heparin or HepS into the reaction mix significantly reduces non-specific interactions of the σ70RNAP and RNAP after RPo formation allowing for better interpretation of complexes shown within AFM images, particularly on DNA templates containing more than one promoter. Previous expectation was that negatively charged polysaccharides, often used as competitive inhibitors of σRNAP binding and RPo formation, would also inhibit binding of the DNA template to the mica support surface and thereby lower the imaging yield of active RNAP-DNA complexes. We found that the reverse of this was true, and that the yield of RPo formation detected by AFM, for a simple tandem gene model containing two λPR promoters, increased. Moreover and unexpectedly, HepS was more efficient than heparin, with both of them having a dispersive effect on the sample, minimising unwanted RNAP-RNAP interactions as well as non-specific interactions between the RNAP and DNA template. The success of this method relied on the observation that E. coli RNAP has the highest affinity for the mica surface of all the molecular components. For our system, the affinity of the three constituent biopolymers to muscovite mica was RNAP>Heparin or HepS>DNA. While we observed that heparin and HepS can inhibit DNA binding to the mica, the presence of E. coli RNAP overcomes this effect allowing a greater yield of RPos for AFM analysis. This method can be extended to other DNA binding proteins and enzymes, which have an affinity to mica higher than DNA, to improve sample preparation for AFM studies.

Single-stranded DNA loops as fiducial markers for exploring DNA-protein interactions in single molecule imaging.

A polymerase chain reaction (PCR) based method of adding a single-stranded DNA (ssDNA) hairpin loop to one end of linear double-stranded (ds) DNA templates was developed. The loop structure serves as a fiducial marker in single molecule imaging by atomic force microscopy (AFM) and can be applied to study DNA-protein interactions. The nucleic acid end-labels allow discrimination of the polarity of the DNA template in the AFM while limiting non-specific interactions which might occur from non-nucleic acid labels. Homo-polynucleotide ssDNA loops made up of 20 base-pairs (bp) for each of the four bases (A, T, G, C) were investigated to determine the effects of sequence on template labelling. The products were produced with high efficiency and high yield with the loop readily distinguished from the dsDNA template by height and diameter in the AFM. The application of the method to study DNA transcription was investigated by firing Escherichia Coli RNA polymerase (RNAP) from a λPR promoter in the direction of the loop-labelled end. The ssDNA loops captured elongating complexes of RNAP, arresting transcription and preventing dissociation. The dual role of the loop as a polarity marker and retainer of previously active RNAP will allow mechanisms of gene expression to be studied with single molecule sensitivity by AFM. This will enable insight into molecular interactions of RNAP on single DNA templates in convergent or tandem transcription configurations.

Single-molecule studies of DNA transcription using atomic force microscopy.

Atomic force microscopy (AFM) can detect single biomacromolecules with a high signal-to-noise ratio on atomically flat biocompatible support surfaces, such as mica. Contrast arises from the innate forces and therefore AFM does not require imaging contrast agents, leading to sample preparation that is relatively straightforward. The ability of AFM to operate in hydrated environments, including humid air and aqueous buffers, allows structure and function of biological and biomolecular systems to be retained. These traits of the AFM are ensuring that it is being increasingly used to study deoxyribonucleic acid (DNA) structure and DNA-protein interactions down to the secondary structure level. This report focuses in particular on reviewing the applications of AFM to the study of DNA transcription in reductionist single-molecule bottom-up approaches. The technique has allowed new insights into the interactions between ribonucleic acid (RNA) polymerase to be gained and enabled quantification of some aspects of the transcription process, such as promoter location, DNA wrapping and elongation. More recently, the trend is towards studying the interactions of more than one enzyme operating on a single DNA template. These methods begin to reveal the mechanics of gene expression at the single-molecule level and will enable us to gain greater understanding of how the genome is transcribed and translated into the proteome.

In vitro studies on erythrosine-based photodynamic therapy of malignant and pre-malignant oral epithelial cells.

Photodynamic Therapy (PDT) involves the administration of a tumor localizing photosensitizing agent, which upon activation with light of an appropriate wavelength leads to the destruction of the tumor cells. The aim of the present study was to determine the efficacy of erythrosine as a photosensitizer for the PDT of oral malignancies. The drug uptake kinetics of erythrosine in malignant (H357) and pre-malignant (DOK) oral epithelial cells and their susceptibility to erythrosine-based PDT was studied along with the determination of the subcellular localization of erythrosine. This was followed by initial investigations into the mechanism of cell killing induced following PDT involving both high and low concentrations of erythrosine. The results showed that at 37 °C the uptake of erythrosine by both DOK and H357 cells increased in an erythrosine dose dependent manner. However, the percentage of cell killing observed following PDT differed between the 2 cell lines; a maximum of ~80% of DOK cell killing was achieved as compared to ~60% killing for H357 cells. Both the DOK and H357 cell types exhibited predominantly mitochondrial accumulation of erythrosine, but the mitochondrial trans-membrane potential (ΔΨ(m)) studies showed that the H357 cells were far more resistant to the changes in ΔΨ(m) when compared to the DOK cells and this might be a factor in the apparent relative resistance of the H357 cells to PDT. Finally, cell death morphology and caspase activity analysis studies demonstrated the occurrence of extensive necrosis with high dose PDT in DOK cells, whereas apoptosis was observed at lower doses of PDT for both cell lines. For H357 cells, high dose PDT produced both apoptotic as well as necrotic responses. This is the first instance of erythrosine-based PDT's usage for cancer cell killing.

Single-stranded loops as end-label polarity markers for double-stranded linear DNA templates in atomic force microscopy.

Visualization of DNA-protein interactions by atomic force microscopy (AFM) has deepened our understanding of molecular processes such as DNA transcription. Interpretation of systems where more than one protein acts on a single template, however, is complicated by protein molecules migrating along the DNA. Single-molecule AFM imaging experiments can reveal more information if the polarity of the template can be determined. A nucleic acid-based approach to end-labelling is desirable because it does not compromise the sample preparation procedures for biomolecular AFM. Here, we report a method involving oligonucleotide loop-primed synthesis for the end labelling of double-stranded DNA to discriminate the polarity of linear templates at the single-molecule level. Single-stranded oligonucleotide primers were designed to allow loop formation while retaining 3'-single-strand extensions to facilitate primer annealing to the template. Following a DNA polymerase extension, the labelled templates were shown to have the ability to form open promoter complexes on a model nested gene template using two Escherichia coli RNA polymerases in a convergent transcription arrangement. Analysis of the AFM images indicates that the added loops have no effect on the ability of the promoters to recruit RNA polymerase. This labelling strategy is proposed as a generic methodology for end-labelling linear DNA for studying DNA-protein interactions by AFM.

Atomic force microscopy of DNA at high humidity: irreversible conformational switching of supercoiled molecules.

Three topologically different double-stranded DNA molecules of the same size (bps) have been imaged in air on mica using amplitude modulation atomic force microscopy (AM AFM) under controlled humidity conditions. At very high relative humidity (>90% RH), localized conformational changes of the DNA were observed, while at lower RH, the molecules remained immobile. The conformational changes occurred irreversibly and were driven principally by superhelical stress stored in the DNA molecules prior to binding to the mica surface. The binding mechanism of the DNA to the mica (surface equilibration versus kinetic trapping) modulated the extent of the conformational changes. In cases where DNA movement was observed, increased kinking of the DNA was seen at high humidity when more surface water was present. Additionally, DNA condensation behavior was also present in localized regions of the molecules. This study illustrates that changes in the tertiary structure of DNA can be induced during AFM imaging at high humidity on mica. We propose that AM AFM in high humidity will be a useful technique for probing DNA topology without some of the drawbacks of imaging under bulk solution.

Possible role of extracellular signal-regulated kinase pathway in regulation of Sox9 mRNA expression in chondrocytes under hydrostatic pressure.

The potential involvement of the extracellular signal-regulated kinase (ERK) pathway in chondrocyte mechanotransduction was tested in bovine chondrocyte-agarose constructs under hydrostatic loading. Results suggested that the ERK pathway may be inhibited by hydrostatic pressure-induced mechanotransduction and may also be a negative regulator of Sox9 mRNA expression, which is an important modulator of chondrocyte function.

Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy.

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 lambda(pr) 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.

Imaging RNA polymerase-amelogenin gene complexes with single molecule resolution using atomic force microscopy.

The AMELX gene encoding the enamel matrix protein, amelogenin, is located within (and in the opposite orientation to) the first intron of the ARHGAP6 gene, which encodes a GTPase-activating protein. The orientation of these two genes with respect to each other raises the possibility that they may undergo simultaneous convergent transcription during amelogenesis. The aim of this study was to use atomic force microscopy (AFM) to study a transcriptionally active amelogenin DNA template and to investigate the binding of RNA polymerase to convergently aligned promoters. Images of RNA polymerases stalled on DNA templates were obtained following incubation of the template with RNA polymerases and ribonucleotide triphosphates. A linear DNA template incorporating an intact rat amelogenin cDNA flanked by convergently aligned coliphage T7 and T3 promoters was constructed and shown to be transcriptionally active in vitro. Atomic force microscopy images of transcription complexes revealed globular structures, corresponding to single RNA polymerase molecules bound at specific locations on the DNA templates. These results indicate that AFM allows the visualization of individual RNA polymerases on DNA templates, offering a realistic approach to investigating the concept of convergent transcription of nested genes, which may lead to an understanding of whether the simultaneous expression of AMELX and ARHGAP6 is possible during the formation of tooth enamel.

Studying silane mobility on hydrated mica using ambient AFM.

The mobility of the mono-functional aminosilane, aminopropyldimethylmethoxysilane (APDMMS), on mica has been investigated under ambient relative humidity (RH) using tapping-mode (TM) AFM. Silane layers were formed from vapour phase at various, controlled humidities and then imaged at ambient laboratory conditions (typically 30-40% RH). At low RH of formation (<25%) films without any resolvable sub-structure were formed, and these were stable to the imaging probe. At high RH of formation (>25%), where islands of phase II water are known to exist on mica, a two-phase domain structure was seen as two height levels and the surface area of the higher domains correlated with the RH. Sequential images taken over a 30-60 min time period show that these domains are mobile and at intermediate to high RH the domains coalesce under the influence of the scanning AFM tip. The results suggest the APDMMS resides at the air-water interface (rather than interacting with the mica) and that it preferentially interacts with the mobile phase II water as opposed to the phase I water tightly bound to the mica.

Formation of aminosilane-functionalized mica for atomic force microscopy imaging of DNA.

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.

Hydrostatic pressure modulates proteoglycan metabolism in chondrocytes seeded in agarose.

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.

Upregulation of aggrecan and type II collagen mRNA expression in bovine chondrocytes by the application of hydrostatic pressure.

The aim of this study was to investigate the effect of hydrostatic pressure on the expression of messenger ribonucleic acid (mRNA) for specific extracellular matrix proteins in chondrocytes. Chondrocytes obtained from bovine metatarsophalangeal joints were embedded in cylindrical 2% agarose gels. A novel experimental system was used to apply 5 MPa of static hydrostatic pressure to these chondrocytes for 4 hours. The application of hydrostatic pressure caused a significant increase in the level of aggrecan mRNA by almost four fold (p<0.01) as well as a 50% increase in the level of type II collagen mRNA (p<0.05). However, there was no significant change in the level of TIMP-1 mRNA. It was suggested that the application of hydrostatic pressure, in the absence of cell deformation, can bring about changes in the matrix components which may play an important role in the homeostasis and mechanical properties of articular cartilage.