Cover image for ChemBioChem

Special Issue: EMBO Symposium: Chemistry Meets Biology

January 7, 2005

Volume 6, Issue 1

Pages 1–206

    1. Cover Picture: Forced Intercalation Probes (FIT Probes): Thiazole Orange as a Fluorescent Base in Peptide Nucleic Acids for Homogeneous Single-Nucleotide-Polymorphism Detection (ChemBioChem 1/2005) (page 1)

      Olaf Köhler, Dilip Venkatrao Jarikote and Oliver Seitz

      Version of Record online: 7 JAN 2005 | DOI: 10.1002/cbic.200590000

      The cover picture shows new PNA probes–FIT Probes–and their use in homogenous DNA detection and was designed by Elke Socher. In FIT Probes, the thiazole orange dye is forced to intercalate at a specific position of a probe–target complex. In their paper on p. 69 ff, O. Seitz et al. describe how the binding of FIT probes with DNA results in fluorescence enhancements due to the coplanarization of the two heterocyclic ring systems. The remarkable attenuation of fluorescence that is observed when forcing thiazole orange to intercalate next to a mismatched base pair allows a DNA target to be distinguished from its single-base mutant under nonstringent hybridization conditions, a property that should be of advantage for real-time quantitative PCR and nucleic acid detection within living cells.

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      Editorial: Closing the Gap between Chemistry and Biology (pages 3–5)

      Orlando Schärer and Carsten Schultz

      Version of Record online: 7 JAN 2005 | DOI: 10.1002/cbic.200400405

    3. Intramers and Aptamers: Applications in Protein-Function Analyses and Potential for Drug Screening (pages 19–26)

      Michael Famulok and Günter Mayer

      Version of Record online: 7 JAN 2005 | DOI: 10.1002/cbic.200400299

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       On target. Aptamers and intramers can be isolated against diverse targets and are used as functional inhibitors to characterise proteins in vitro or in living organisms. As nucleic acids, they are predestined for the functional validation of intracellular proteins or protein subdomains. Recent developments have boosted aptamer technology for applications such as small-molecule screening (see scheme); this underlines the fact that aptamers provide an exciting novel interface between target validation and drug development.

    4. DNA Interstrand Crosslinks: Natural and Drug-Induced DNA Adducts that Induce Unique Cellular Responses (pages 27–32)

      Orlando D. Schärer

      Version of Record online: 7 JAN 2005 | DOI: 10.1002/cbic.200400287

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       Tied down. DNA interstrand crosslinks are cytotoxic adducts formed naturally and by drugs used in antitumor therapy. Our current knowledge of the cellular responses induced by crosslinks and new methods for the synthesis of crosslinks developed to study the biological responses they induce are reviewed.

    5. Small-Molecule Screening and Profiling by Using Automated Microscopy (pages 33–39)

      Timothy J. Mitchison

      Version of Record online: 29 NOV 2004 | DOI: 10.1002/cbic.200400272

       All together now. Small molecules with known bioactivity have been profiled by using multiple fluorescent probes, and clustered into mechanistic classes by automated statistical analysis, which allows large data sets of cell images to be converted into numbers that can be used to score for a desired effect. The high information content of automated microscopy and the potential for discovering new targets and ligands make the technology worth considering. It should be especially useful for relating biochemical activity to phenotypic effects in programs in which specificity is a challenge, such as kinase and histone deacetylase inhibitors. Cytological profiling combined with other multidimensional profiling methods should help solve the difficult problem of predicting the biological effect of drugs prior to clinical trails, and thus lower the time and cost of therapeutic drug discovery.

    6. Chemical Approaches to Controlling Intracellular Protein Degradation (pages 40–46)

      John S. Schneekloth Jr. and Craig M. Crews

      Version of Record online: 8 NOV 2004 | DOI: 10.1002/cbic.200400274

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      Inactive. Recent advances have yielded many ways to study proteins by means of inactivation. Traditional methods of genetic knockout are complimented by newer techniques, including RNAi and small molecules that induce proteolysis (see scheme). Although seemingly in competition, these techniques each offer solutions to specific problems in proteomic analysis.

    7. Protein Chemistry on the Surface of Living Cells (pages 47–52)

      Nils Johnsson, Nathalie George and Kai Johnsson

      Version of Record online: 19 NOV 2004 | DOI: 10.1002/cbic.200400290

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      Promiscuous, yet specific. A general method for the covalent labeling of the cell-surface fusion proteins of acyl carrier proteins with chemically diverse molecules (see formula) is presented. The labeling is highly specific with respect to the fusion protein (see fluorescence micrograph) but promiscuous with respect to the nature of the label. These features will allow the approach to become an important tool for studying cell-surface proteins and for complementing existing approaches in cellsurface engineering.

    8. Direct Quantitation of Poly(ADP-Ribose) Polymerase (PARP) Activity as a Means to Distinguish Necrotic and Apoptotic Death in Cell and Tissue Samples (pages 53–55)

      Karson S. Putt, Gregory J. Beilman and Paul J. Hergenrother

      Version of Record online: 17 NOV 2004 | DOI: 10.1002/cbic.200400330

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      PARP-angel of death. The PARP-1 enzyme is either inactivated or activated depending on whether a cell dies via apoptosis or necrosis (see scheme). This differential processing of PARP-1 can now be used as a rapid, sensitive, and convenient method to distinguish apoptotic from necrotic cell death, and is even applied to quantitate necrosis in bulk tissue from a pig in which a hemorrhagic shock was induced.

    9. Site-Specific in vivo Labeling of Proteins for NMR Studies (pages 55–58)

      Alexander Deiters, Bernhard H. Geierstanger and Peter G. Schultz

      Version of Record online: 17 NOV 2004 | DOI: 10.1002/cbic.200400319

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      Unnatural amino acid mutagenesis was used for the site-specific incorporation of an isotopically labeled amino acid, shown here, into a protein in vivo, and protein amounts sufficient for NMR studies were produced.

    10. Aminoglycoside-Hybrid Ligands Targeting the Ribosomal Decoding Site (pages 58–65)

      Dionisios Vourloumis, Geoffrey C. Winters, Klaus B. Simonsen, Masayuki Takahashi, Benjamin K. Ayida, Sarah Shandrick, Qiang Zhao, Qing Han and Thomas Hermann

      Version of Record online: 29 NOV 2004 | DOI: 10.1002/cbic.200400197

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      Bacterial bridges: RNA helix 44 within the small subunit of the bacterial ribosome is the target for natural aminoglycoside antibiotics of the neomycin and hygromycin classes, which bind at neighboring sites and thereby interfere with protein synthesis. We describe synthetic aminoglycoside-hybrid ligands, such as 1, designed to inhibit translation by bridging between the neamine and hygromycin binding sites.

    11. Extended Target Sequence Specificity of PNA–Minor-Groove Binder Conjugates (pages 66–68)

      Peter E. Nielsen, Karin Frederiksen and Carsten Behrens

      Version of Record online: 9 DEC 2004 | DOI: 10.1002/cbic.200400251

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       Conjugal bliss. We show that a peptide nucleic acid–Hoechst conjugate (see scheme) kinetically and sequence preferentially guides the PNA moiety to target a binding site proximal to an A–T region that has an affinity for the minor-groove binder. These results demonstrate a new strategy for constructing DNA recognition ligands composed of a sequence-guiding domain that increases the target specificity and a DNA-modification domain that determines the biological activity of the conjugate.

    12. Forced Intercalation Probes (FIT Probes): Thiazole Orange as a Fluorescent Base in Peptide Nucleic Acids for Homogeneous Single-Nucleotide-Polymorphism Detection (pages 69–77)

      Olaf Köhler, Dilip Venkatrao Jarikote and Oliver Seitz

      Version of Record online: 7 DEC 2004 | DOI: 10.1002/cbic.200400260

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       FIT for the job: New peptide nucleic acid–based probes are described in which thiazole orange (TO) dyes serve as fluorescent base surrogates (see structure; B=A, C, G, or T). The identity of the linker that connects the fluorophore with the PNA backbone decides whether a surrogate conveys sequence-selective DNA binding and whether fluorescence is increased or decreased upon single-mismatched hybridization. The best-suited TO derivative enabled a DNA target to be distinguished from its single-base mutant even under nonstringent hybridization conditions.

    13. Imaging Activation of Two Ras Isoforms Simultaneously in a Single Cell (pages 78–85)

      Anna Peyker, Oliver Rocks and Philippe I. H. Bastiaens

      Version of Record online: 7 JAN 2005 | DOI: 10.1002/cbic.200400280

       Imaging multiple protein kinetics. To investigate the spatiotemporal organization of a cellular network it is necessary to correlate protein activities within that network. A new FRET-based method has been introduced to measure the activation kinetics of two proteins in the same cell. Using this approach, we demonstrate a localization-dependent regulation of Ras activity at the plasma membrane and at the Golgi.

    14. Synthesis and Application of Fluorescent Ras Proteins for Live-Cell Imaging (pages 86–94)

      Reinhard Reents, Melanie Wagner, Stefanie Schlummer, Jürgen Kuhlmann and Herbert Waldmann

      Version of Record online: 7 JAN 2005 | DOI: 10.1002/cbic.200400233

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      Ras lights up: Semisynthetic Ras proteins are efficient probes for cell-biology experiments. With a fluorophore introduced at an appropriate site these proteins are processed by the cellular machinery and their intracellular localization can then be visualized by means of confocal laser fluorescence microscopy.

    15. Affinity Chromatography of Tryptases: Design, Synthesis and Characterization of a Novel Matrix-Bound Bivalent Inhibitor (pages 95–103)

      Norbert Schaschke, Dusica Gabrijelcic-Geiger, Andreas Dominik and Christian P. Sommerhoff

      Version of Record online: 9 DEC 2004 | DOI: 10.1002/cbic.200400217

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      Their tetrameric architecture makes human tryptases unique among trypsin-like serine proteases. An immobilized bivalent ligand takes advantage of this feature for the selective recognition and binding of β-tryptase from a crude protein mixture and therefore provides a novel geometry-driven approach to isolation and purification of human mast cell tryptases.

    16. Role of DNA Sequence in the Binding Specificity of Synthetic Basic-Helix-Loop-Helix Domains (pages 104–113)

      Amy C. Beltran, Philip E. Dawson and Joel M. Gottesfeld

      Version of Record online: 9 DEC 2004 | DOI: 10.1002/cbic.200400184

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      Beyond the box. DNA bases outside of the consensus E-box sequence play an important role in determining binding affinities and specificities for members of the basic helix-loop-helix (bHLH) class of transcription factors. Access to synthetic DNA and protein enabled the analysis of specific interactions between a conserved arginine residue, shown in the picture, in the basic helix of each bHLH domain and adenine in a flanking position two bases 5′ to the E-box.

    17. Direct Observation of Anion-Mediated Translocation of Fluorescent Oligoarginine Carriers into and across Bulk Liquid and Anionic Bilayer Membranes (pages 114–122)

      Naomi Sakai, Toshihide Takeuchi, Shiroh Futaki and Stefan Matile

      Version of Record online: 17 NOV 2004 | DOI: 10.1002/cbic.200400256

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      Dissection into isolate processes is the key to learning how counteranions mediate each step of the translocation of cell-penetrating peptides like oligoarginines across intact lipid-bilayer membranes (see picture).

    18. Porphyrin Derivatives for Telomere Binding and Telomerase Inhibition (pages 123–132)

      Isabelle M. Dixon, Frédéric Lopez, Jean-Pierre Estève, Agueda M. Tejera, María A. Blasco, Geneviève Pratviel and Bernard Meunier

      Version of Record online: 18 NOV 2004 | DOI: 10.1002/cbic.200400113

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      Depending on the nature of the coordinated ion, cationic metalloporphyrins show different modes and kinetics of interaction with G-quadruplex DNA. SPR studies show that molecules able to stack with the last G-tetrad give fast interactions while external binders interact more slowly and selectively (see diagram).

    19. A New Family of Synthetic Diterpenes that Regulates Cytokine Synthesis by Inhibiting IκBα Phosphorylation (pages 133–144)

      Ta-Hsiang Chao, Thanh Lam, Binh G. Vong, Paqui G. Través, Sonsoles Hortelano, Chinmay Chowdhury, F. Rena Bahjat, G. Kenneth Lloyd, Lyle L. Moldawer, Lisardo Boscá, Michael A. Palladino and Emmanuel A. Theodorakis

      Version of Record online: 12 NOV 2004 | DOI: 10.1002/cbic.200400089

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      Not all puffed up. The privileged structure of acanthoic acid (1) led to the synthesis of a new family of synthetic diterpenes, represented by motif 2, that have potent anti-inflammatory activities. The mode of action of these compounds may involve inhibition of IκBα phosphorylation, thereby regulating the NF-κB-mediated synthesis of certain proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6.

    20. High-Content Screening and Profiling of Drug Activity in an Automated Centrosome-Duplication Assay (pages 145–151)

      Zachary E. Perlman, Timothy J. Mitchison and Thomas U. Mayer

      Version of Record online: 29 NOV 2004 | DOI: 10.1002/cbic.200400266

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       Spotting the spots. Regulation of centrosome duplication may be a key pathway that affects the onset of cancer. Using a cytometric profiling approach, we have screened libraries of known drugs to suggest unexpected effects by PKC inhibitors and retinoic acid agonists, and identified a number of small molecules that inhibit centrosome duplication by apparently new mechanisms.

    21. Chemical Inhibitors when Timing Is Critical: A Pharmacological Concept for the Maturation of T Cell Contacts (pages 152–161)

      Karsten Köhler, Annemarie C. Lellouch, Susanne Vollmer, Oda Stoevesandt, Antje Hoff, Lasse Peters, Hans Rogl, Bernard Malissen and Roland Brock

      Version of Record online: 7 JAN 2005 | DOI: 10.1002/cbic.200400241

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       The timing of signal-transduction events critically determines a cellular response. Small-molecule inhibitors enable the analysis of the function of a gene product at any point in time during a signalling cascade. By using the Lck tyrosine kinase inhibitor PP2, we show that 5 min after contact formation, the activity of this enzyme is required for the maintenance of T cell receptor contacts. Contacts are Lck independent at later time points.

    22. The Transcriptional Coactivator p300 Plays a Critical Role in the Hypertrophic and Protective Pathways Induced by Phenylephrine in Cardiac Cells but Is Specific to the Hypertrophic Effect of Urocortin (pages 162–170)

      Sean M. Davidson, Paul A. Townsend, Chris Carroll, Alexander Yurek-George, Karanam Balasubramanyam, Tapas K. Kundu, Anastasis Stephanou, Graham Packham, A. Ganesan and David S. Latchman

      Version of Record online: 9 DEC 2004 | DOI: 10.1002/cbic.200400246

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      Healing hearts. Histone acetyl-transferases (HATs) and histone deacetylases (HDACs) have opposing actions. However, anacardic acid, an inhibitor of HAT activity, and spiruchostatin A, an inhibitor of HDACs were both found to prevent cardiomyocyte hypertrophy. (A typical hypertrophic cell is shown.) A possible explanation involving HDAC isoform specificity is offered.

    23. Aristoforin, a Novel Stable Derivative of Hyperforin, Is a Potent Anticancer Agent (pages 171–177)

      Michael Gartner, Thomas Müller, Jan C. Simon, Athanassios Giannis and Jonathan P. Sleeman

      Version of Record online: 9 DEC 2004 | DOI: 10.1002/cbic.200400195

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      Problem solved. Hyperforin (1), a natural product of St. John's wort, has antitumor properties in vitro and in vivo but it is highly unstable and poorly soluble in aqueous solution. The synthesis of semisynthetic hyperforin derivatives led to compounds with improved stability and solubility compared to hyperforin. Moreover, one of these compounds, Aristoforin (2), has antitumor properties both in vitro and in vivo to a similar extent to hyperforin; this suggests it has potential as a novel anticancer drug.

    24. Site-Specific Cleavage—A Model System for the Identification of Lipid-Modified Glutamate Residues in Proteins (pages 178–185)

      Peter Sawatzki, Ingo Damm, Barbara Pierstorff, Heike Hupfer, Konrad Sandhoff and Thomas Kolter

      Version of Record online: 9 DEC 2004 | DOI: 10.1002/cbic.200400189

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      Basic chemistry. A site-specific reaction for the determination of glutamic acid ester modifications in peptides based on the use of base-labile acylpyrrolidine-2-ones has been developed.

    25. Synthesis, Inhibition Properties, and Theoretical Study of the New Nanomolar Trehalase Inhibitor 1-Thiatrehazolin: Towards a Structural Understanding of Trehazolin Inhibition (pages 186–191)

      Jose Luis Chiara, Isabel Storch de Gracia, Ángela García, Ágatha Bastida, Sofía Bobo and María D. Martín-Ortega

      Version of Record online: 8 NOV 2004 | DOI: 10.1002/cbic.200400231

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      Differences in the trehalase-inhibition properties of trehazolin and its new and potent analogue 1-thiatrehazolin are correctly predicted by ab initio model calculations of the bidentate interaction shown.

    26. Oligonucleotide Bearing Ethylenediamine-N,N,N′-Triacetates for Gap-Selective DNA Hydrolysis by Ce4+/EDTA (pages 192–196)

      Makoto Komiyama, Hiroyuki Arishima, Masahito Yokoyama, Yoshihito Kitamura and Yoji Yamamoto

      Version of Record online: 12 NOV 2004 | DOI: 10.1002/cbic.200400220

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      By using DNA additives bearing ethylenediamine-N,N,N′-triacetate groups, a gap structure was formed in substrate DNA so that the ligand groups (X) were placed at both edges of the gap. The target site was site-selectively hydrolyzed by a CeIV/EDTA complex.

    27. Hoechst 33258 as a pH-Sensitive Probe to Study the Interaction of Amine Oxide Surfactants with DNA (pages 197–203)

      Laura Goracci, Raimondo Germani, Gianfranco Savelli and Dario M. Bassani

      Version of Record online: 17 NOV 2004 | DOI: 10.1002/cbic.200400196

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      Acid test. The use of Hoechst 33258 as a fluorescent probe to characterize the interaction between DNA and pH-sensitive amphiphiles such as 1 is discussed. The findings support the intriguing possibility that Hoechst 33258 can “read” local pH variations in the proximity of the DNA–solution interface that occur upon surfactant binding. The graph shows the change in fluorescent emission at pH 5.8 with various concentrations of 1.

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      Preview: ChemBioChem 1/2005 (page 206)

      Version of Record online: 7 JAN 2005 | DOI: 10.1002/cbic.200590001