Cooperative strand displacement by peptide nucleic acid (PNA).

BACKGROUND: Synthetic homopyrimidine peptide nucleic acids (PNAs) can bind complementary targets in double-stranded DNA, generating strand-displacement complexes, and so offering an opportunity to modulate specific gene expression. Several issues remain to be addressed before these attributes can be exploited in vivo, however. RESULTS: The kinetics of the interaction between a homopyrimidine PNA and a complementary homopurine target on double-stranded DNA were analyzed in the presence or absence of a preformed strand-displacement complex proximal to the target. The complex was established under low salt conditions by the binding of a different homopyrimidine PNA to a target situated adjacent to the first PNA target. These two targets were placed next to each other on opposite strands at distances of 0, 2, 4 and 8 base pairs apart. The presence of a preformed strand-displacement complex near the target accelerates the binding of PNA to double-stranded DNA in a salt-dependent manner. The influence of salt on the binding rates was also examined. The binding rate is increased by a factor of 1 x exp(70[NaCl]), that is, 16-fold at 40 mM NaCl and more than 10(4)-fold if extrapolated to 140 mM NaCl. This effect is significantly reduced if the two targets are 2 base pairs apart and completely absent if the distance is 4 base pairs or more. CONCLUSIONS: The perturbation of the DNA helix imposed by a PNA strand-displacement complex only propagates a few base pairs. It is therefore possible to target sites in the immediate vicinity of strand invasion complexes specifically. The results presented have implications for the mechanism of strand displacement and for the application of PNA in a genomic context.

Interaction of checkpoint kinase 1 and the X-linked inhibitor of apoptosis during mitosis.

We report here that the checkpoint kinase Chk1 and the inhibitor of apoptosis protein (IAP) family member XIAP can be found in a complex in association with condensed chromosomes aligned at the metaphase plate during mitosis. The interaction between Chk1 and XIAP was transient and followed the breakdown of the nuclear envelope. Chk1 and XIAP also formed a complex in vitro and in coimmunoprecipitation experiments. The interaction between Chk1 and the BIR3 domain of XIAP in vitro required an N-terminal sequence in Chk1 that is identical to the BIR-binding motif at the N-terminus of HID. An interaction of Chk1 and XIAP may imply a mechanism of coupling between the regulatory networks that control cell cycle progression and apoptosis during mitosis.

Convergent evolution with combinatorial peptides.

Once the sequence of a genome is in hand, understanding the function of its encoded proteins becomes a task of paramount importance. Much like the biochemists who first outlined different biochemical pathways, many genomic scientists are engaged in determining which proteins interact with which proteins, thereby establishing a protein interaction network. While these interactions have evolved in regard to their specificity, affinity and cellular function over billions of years, it is possible in the laboratory to isolate peptides from combinatorial libraries that bind to the same proteins with similar specificity, affinity and primary structures, which resemble those of the natural interacting proteins. We have termed this phenomenon 'convergent evolution'. In this review, we highlight various examples of convergent evolution that have been uncovered in experiments dissecting protein-protein interactions with combinatorial peptides. Thus, a fruitful approach for mapping protein-protein interactions is to isolate peptide ligands to a target protein and identify candidate interacting proteins in a sequenced genome by computer analysis.

The complexity of TNF-related apoptosis-inducing ligand.

One of the major goals of researchers in the field of apoptosis is to understand the molecular mechanisms of the various components of the apoptotic pathways, with the hope to identify targets for novel cancer therapies. The discovery of a TNF-related, apoptosis-inducing ligand, TRAIL, that kills transformed cells with great specificity in vitro, has provided the hope that TRAIL may be used to induce cell death in tumor cells without affecting normal tissues. However, TRAIL signaling is very complex and a clear understanding of its function is necessary before it can be used in cancer therapy. Complexity of TRAIL-induced signaling is apparent from its ubiquitous expression, its ability to interact with five receptors, and its tumor-selective induction of apoptosis. The signaling events that mediate the tumor selectivity of TRAIL-induced apoptosis and the biological functions of each of the TRAIL receptors are not well characterized. This review will focus on the complexity of TRAIL and the role of c-FLIP in mediating TRAIL function.

Testing the quality of electron microscope mapping data for DNA molecules with sequence-specific ligands.

A procedure for the testing of Electron Microscope (EM) mapping data for DNA molecules with site-specific bound ligands is suggested. The difficulty of distinguishing DNA molecule ends on electron micrographs indicates that their true orientations are not known. This in turn presents problems in obtaining correct maps relating to their alignment, and complicates checking the maps' validity. For these reasons a computer simulation of the EM study of double-stranded DNA molecules with site-specific bound ligands was carried out. The knowledge of the true orientations of the simulated DNA molecules allowed us to examine their final orientations after alignment. We used the number of improper-oriented molecules as the quantitative measure of the map quality. Detailed investigation based on this parameter permitted us to invent the criterion for the map validity, and to suggest the procedure for the testing of alignment of real DNA molecules. This procedure implies multiple randomization of initial orientations of the DNA molecules and minute analysis of the final maps. Most of the molecular, statistical and experimental parameters inherent to EM investigation of site-specific binding, such as the number of specific binding sites (N), the mean number of bound ligands (A), the length of the DNA molecules (L), the specific/non-specific ratio of binding (K), together with the standard deviation of DNA molecule lengths (HL) were tested for their influence upon the quality of EM mapping data. An empirical equation for the ultimate values of these parameters has been found, allowing us to predict the success of EM mapping.

[Etiology of inflammatory maxillofacial diseases and estimation of the effectiveness of antibacterial drugs in vitro]. / Etiologiia vospalitel'nykh zabolevanii cheliustno-litsevoi oblasti i otsenka éffektivnosti antibakterial'nykh preparatov in vitro.

The results of identification of 710 clinical strains of anaerobic microorganisms isolated from the pathological foci of patients with maxillofacial diseases are presented. The species composition of the microflora associations in the cases with abscesses, phlegmon, lymphadenitis, osteomyelitis and parodontitis is described. Along with a high frequency of nonsporulating anaerobes, staphylococci, microaerophilic streptococci and in the cases with parodontitis actinomycetes, Bacillus licheniformis and Bacillus coagulans strains (1.6-15% of the isolated strains) were first detected in cases with various forms of the disease. Two groups of the drugs effective against the anaerobes were identified by the data on the antibiotic sensitivity. The lowest MICs along with the activity broad spectrum were defined for gramicidin, levomycetin and nitazol.

[The possibilities of using clinical blood analysis for the prognosis of the course of acute suppurative inflammatory processes in the soft tissues of the maxillofacial area]. / Vozmozhnosti ispol'zovaniia klinicheskogo analiza krovi dlia prognozirovaniia techeniia ostrykh gnoinykh vospalitel'nykh protsessov miagkikh tkanei cheliustno-litsevoi oblasti.

The authors have attempted to mathematically predict the length of inpatient treatment of patients with maxillofacial acute pyoinflammatory processes basing on the regression analysis of the peripheral blood parameters. A number of mathematical formulae fit for wide clinical application were derived.

[Relationship between nonspecific, immunologic reactivity of the body and the type of course of an acute inflammatory process]. / Sviaz' mezhdu nespetsificheskoi, immunologicheskoi reaktivnost'iu organizma i tipom techeniia ostrogo vospalitel'nogo protsessa.

By using the results of clinical, laboratory, and immunological studies, the authors show that the type (normoergic, hypoergic, hyperergic) of an inflammatory response is related to the parameters of nonspecific and immunological responsiveness of patients with pyo-inflammation in the maxillofacial region. The hypoergic inflammatory response is characterized by the least responsiveness mainly to its cellular link. The hyperergic inflammatory reaction is chiefly caused by considerably enhanced phagocytosis.

Molecular recognition properties of the C-terminal Sh3 domain of the Cbl associated protein, Cap.

A phage-displayed combinatorial peptide library was used to define the specificity of one of the three Src homology 3 (SH3) domains in a novel cytoskeletal protein, named CAP, for Cbl Associated Protein. The C-terminal SH3 domain was used to affinity select peptides with the consensus, PXPPXRXSSL, from a library of X6PXXPX6 peptides. Peptide sequences resembling this consensus were identified in two signal transduction proteins, c-Cbl and son-on-sevenless (Sos), previously shown to interact with the C-terminal SH3 domain of CAP. Genetic fusion of 16 and 14 amino acid segments of c-Cbl and Sos, respectively, to bacterial alkaline phosphatase confirmed that these segments were potential ligand sites for the C-terminal SH3 domain of CAP. Alanine-scanning mutagenesis of the c-Cbl peptide ligand confirmed that most of the residues, which were conserved among the peptide ligands selected from the combinatorial peptide library, contributed to binding to the C-terminal SH3 domain of CAP.

Cyclic peptides as non-carboxyl-terminal ligands of syntrophin PDZ domains.

Syntrophins, a family of intracellular peripheral membrane proteins of the dystrophin-associated protein complex (DAPC), each contain a single PDZ domain. Syntrophin PDZ domains bind C-terminal peptide sequences with the consensus R/K-E-S/T-X-V-COOH, an interaction that mediates association of skeletal muscle sodium channels with the DAPC. Here, we have isolated cyclic peptide ligands for syntrophin PDZ domains from a library of combinatorial peptides displayed at the N terminus of protein III of bacteriophage M13. Affinity selection from a library of X10C peptides yielded ligands with the consensus X-(R/K)-E-T-C-L/M-A-G-X-Psi-C, where Psi represents any hydrophobic amino acid. These peptides contain residues (underlined) similar to the C-terminal consensus sequence for binding to syntrophin PDZ domains and bind to the same site on syntrophin PDZ domains as C-terminal peptides, but do not bind to other closely related PDZ domains. PDZ binding is dependent on the formation of an intramolecular disulfide bond in the peptides, since treatment with dithiothreitol, or substitution of either of the two cysteines with alanines, eliminated this activity. Furthermore, amino acid replacements revealed that most residues in the phage-selected peptides are required for binding. Our results define a new mode of binding to PDZ domains and suggest that proteins containing similar conformationally constrained sequences may be ligands for PDZ domains.