A novel family of RGD-containing disintegrins (Tablysin-15) from the salivary gland of the horsefly Tabanus yao targets αIIbß3 or αVß3 and inhibits platelet aggregation and angiogenesis.

A novel family of RGD-containing molecules (Tablysin-15) has been molecularly characterised from the salivary gland of the haematophagous horsefly Tabanus yao. Tablysin-15 does not share primary sequence homology to any disintegrin discovered so far, and displays an RGD motif in the N-terminus of the molecule. It is also distinct from disintegrins from Viperidae since its mature form is not released from a metalloproteinase precursor. Tablysin-15 exhibits high affinity binding for platelet αIIbß3 and endothelial cell αVß3 integrins, but not for α5ß1 or α2ß1. Accordingly, it blocks endothelial cell adhesion to vitronectin (IC50 ~1 nM) and marginally to fibronectin (IC50 ~1 µM), but not to collagen. It also inhibits fibroblast growth factor (FGF)-induced endothelial cell proliferation, and attenuates tube formation in vitro. In platelets, Tablysin-15 inhibits aggregation induced by collagen, ADP and convulxin, and prevents static platelet adhesion to immobilised fibrinogen. In addition, solid-phase assays and flow cytometry demonstrates that αIIbß3 binds to Tablysin-15. Moreover, immobilised Tablysin-15 supports platelet adhesion by a mechanism which was blocked by anti-integrin αIIbß3 monoclonal antibody (e.g. abciximab) or by EDTA. Furthermore, Tablysin-15 dose-dependently attenuates thrombus formation to collagen under flow. Consistent with these findings, Tablysin-15 displays antithrombotic properties in vivo suggesting that it is a useful tool to block αIIbß3, or as a prototype to develop antithrombotics. The RGD motif in the unique sequence of Tablysin-15 represents a novel template for studying the structure-function relationship of the disintegrin family of inhibitors.

Development and application of quantum dots for immunocytochemistry of human erythrocytes.

Recent developments in quantum dot technology have resulted in the introduction of new fluorescence immunocytochemical probes. In contrast to organic fluorophores, which are not photostable, the high quantum yield and remarkable photostability of quantum dots solve major problems associated with immunocytochemical studies of erythrocytes. We report here the first application of quantum dots to immunocytochemical studies of human erythrocytes capable of being used in high-magnification, three-dimensional erythrocyte reconstruction techniques. The procedure consists of stabilizing human erythrocytes with a homofunctional imidoester cross-linker to minimize fixative-induced autofluorescence followed by reacting with a quantum dot - monoclonal antibody complex to label band 3 protein. Our new procedure clearly showed a non-homogeneous, raft-like distribution of band 3 protein in the erythrocyte membrane. We also demonstrate the applicability of our technique to studies of erythrocyte membrane modifications occurring during the invasion of a malaria parasite.

Atomic force microscopy sees nucleosome positioning and histone H1-induced compaction in reconstituted chromatin.

We addressed the question of how nuclear histones and DNA interact and form a nucleosome structure by applying atomic force microscopy to an in vitro reconstituted chromatin system. The molecular images obtained by atomic force microscopy demonstrated that oligonucleosomes reconstituted with purified core histones and DNA yielded a 'beads on a string' structure with each nucleosome trapping 158 +/- 27 bp DNA. When dinucleosomes were assembled on a DNA fragment containing two tandem repeats of the positioning sequence of the Xenopus 5S RNA gene, two nucleosomes were located around each positioning sequence. The spacing of the nucleosomes fluctuated in the absence of salt and the nucleosomes were stabilized around the range of the positioning signals in the presence of 50 mM NaCl. An addition of histone H1 to the system resulted in a tight compaction of the dinucleosomal structure.

Long range interaction of cis-DNA elements mediated by architectural transcription factor Bach1.

BACKGROUND: A central question in vertebrate transcriptional regulation is how cis-regulatory modules, including enhancers, silencers and promoters, communicate with each other over long distances to mandate proper gene expression. In order to address this question we analysed protein/DNA interactions in the human beta-globin locus control region (LCR). One of the many proteins that are potentially implicated in LCR function is Bach1. Bach1 possesses a basic leucine zipper (bZip) domain, as well as a BTB/POZ domain that has been shown to be involved in the regulation of chromatin structure. Bach1 forms heterodimers with small Maf proteins through its leucine zipper and binds to Maf recognition elements (MARE). RESULTS: Using atomic force microscopy we visualized large looped DNA structures between MAREs located in different regulatory elements within the human beta-globin LCR that were mediated by Bach1/MafK heterodimers. The formation of these DNA loops required the Bach1 BTB/POZ protein interaction domain. Furthermore, in transfection studies we found that Bach1 repressed the enhancer activity of the LCR in a BTB/POZ domain-dependent manner. CONCLUSION: Our results suggest that Bach1 and other BTB/POZ transcription factors may represent a class of nuclear architectural proteins that mediate long range interactions between cis-regulatory elements in order to regulate gene expression.

Quantitative analysis of the transcription factor AP2 binding to DNA by atomic force microscopy.

Atomic force microscopy (AFM) allows to study the molecular structure of biological macromolecules with nm to A resolutions without crystallization. We show here the applicability of AFM in the quantitative analysis of the molecular mechanisms of DNA/protein interaction: (i) Protein-binding sites can be mapped over a few kilobases of target DNA. (ii) Multimerization state of DNA-binding proteins can be determined simply by measuring the sizes of proteins bound to the DNA. These features are significant advantages over the capabilities provided by conventional techniques in biochemistry and molecular and structural biology.

Atomic force microscopy proposes a novel model for stem-loop structure that binds a heat shock protein in the Staphylococcus aureus HSP70 operon.

The Staphylococcus aureus HSP70 operon produces a polycistronic RNA in response to heat shock, and ORF37 is the first protein to be translated. The promoter of this operon contains a palindromic nucleotide sequence that may form a stem-loop structure. Structural analysis of the promoter regions by atomic force microscopy (AFM) revealed a quadruplet that consists of a pair of stem-loops. A novel "SL2S' (Stem-Loop-Loop-Stem) model was proposed for this structure. AFM also revealed the binding of ORF37 to the quadruplet, establishing a molecular mechanism for this heat shock gene expression; ORF37 acts as a regulator by binding to the SL2S structure in the promoter.

Molecular imaging of Escherichia coli F0F1-ATPase in reconstituted membranes using atomic force microscopy.

The structure of Escherichia coli F0F1-ATPase (ATP synthase), and its F0 sector reconstituted in lipid membranes was analyzed using atomic force microscopy (AFM) by tapping-mode operation. The majority of F0F1-ATPases were visualized as spheres with a calculated diameter of approximately 90 angstroms, and a height of approximately 100 angstroms from the membrane surface. F0 sectors were visualized as two different ring-like structures (one with a central mass and the other with a central hollow of greater than or equal to 18 angstroms depth) with a calculated outer diameter of approximately 130 angstroms. The two different images possibly represent the opposite orientations of the complex in the membranes. The ring-like projections of both images suggest inherently asymmetric assemblies of the subunits in the F0 sector. Considering the stoichiometry of F0 subunits, the area of the image observed is large enough to accommodate all three F0 subunits in an asymmetric manner.