bluuebunny/crossref_metadata_2025 huggingface dataset:
Link, twist, energy, and the stability of DNA minicircles
Authors: Hoffman Kathleen A. , Manning Robert S. , Maddocks John H.
Published: September 2003

We describe how the stability properties of DNA minicircles can be directly read from plots of various biologically intuitive quantities along families of equilibrium configurations. Our conclusions follow from extensions of the mathematical theory of distinguished bifurcation diagrams9, 21 that are applied within the specific context of an elastic rod model of minicircles. Families of equilibria arise as a twisting angle α is varied. This angle is intimately related to the continuously varying linking number Lk for nicked DNA configurations that is defined as the sum of Twist and Writhe. We present several examples of such distinguished bifurcation diagrams involving plots of the energy E, linking number Lk, and a twist moment m3, along families of cyclized equilibria of both intrinsically straight and intrinsically curved DNA fragments. © 2003 Wiley Periodicals, Inc. Biopolymers 70: 145–157, 2003
Structure of an elastin‐mimetic polypeptide by solid‐state NMR chemical shift analysis
Authors: Hong M. , Isailovic D. , McMillan R.A. , Conticello V.P.
Published: September 2003

The conformation of an elastin‐mimetic recombinant protein, [(VPGVG)4(VPGKG)]39, is investigated using solid‐state NMR spectroscopy. The protein is extensively labeled with 13C and 15N, and two‐dimensional 13C‐13C and 15N‐13C correlation experiments were carried out to resolve and assign the isotropic chemical shifts of the various sites. The Pro 15N, 13Cα, and 13Cβ isotropic shifts, and the Gly‐3 Cα isotropic and anisotropic chemical shifts support the predominance of type‐II β‐turn structure at the Pro‐Gly pair but reject a type‐I β‐turn. The Val‐1 preceding Pro adopts mostly β‐sheet torsion angles, while the Val‐4 chemical shifts are intermediate between those of helix and sheet. The protein exhibits a significant conformational distribution, shown by the broad line widths of the 15N and 13C spectra. The average chemical shifts of the solid protein are similar to the values in solution, suggesting that the low‐hydration polypeptide maintains the same conformation as in solution. The ability to measure these conformational restraints by solid‐state NMR opens the possibility of determining the detailed structure of this class of fibrous proteins through torsion angles and distances. © 2003 Wiley Periodicals, Inc. Biopolymers 70: 158–168, 2003
Hyaluronate tetrasaccharide‐ CU(II) interaction: A NMR study
Authors: D'Auria Gabriella , Flores Germana , Falcigno Lucia , Oliva Romina , Vacatello Manuela , Michela Corsaro Maria , Parrilli Michelangelo , Paolillo Livio
Published: September 2003

The coordination of Cu(II) to a hyaluronate tetrasaccharide (HAt) was investigated in aqueous solution by 13C and 1H relaxation measurements at two magnetic fields, 9 and 14 T. The HAt interaction with the metal ion was monitored following the nuclear paramagnetic relaxation enhancements R1p and R2p produced by the copper addition. The data analysis shows that the paramagnetic effect is differently experienced by the nuclei in different monosaccharide residues. A molecular model for the complex HAt‐Cu(II) was built taking into account the experimental data. The model shows the presence of two binding sites, both involving the carboxylate groups of the two glucuronic acid units. The first site, that best simulates the HA binding site, is located on the ligand core, while the second one is located on the terminal glucuronic acid residue. Both binding sites involve, in addition to the carboxylate groups, the O4 oxygens of the glucuronic acid residues. © 2003 Wiley Periodicals, Inc. Biopolymers 70: 260–269, 2003
An assessment of protein–ligand binding site polarizability
Authors: Nayeem Akbar , Krystek Stanley , Stouch Terry
Published: September 2003

Electronic polarizability, an important physical property of biomolecules, is currently ignored in most biomolecular calculations. Yet, it is widely believed that polarization could account for a substantial fraction of the total nonbonded energy of a system. This belief is supported by studies of small complexes in vacuum. This perception is driving the development of a new class of polarizable force fields for biomolecular calculations. However, the quantification of this term for protein–ligand complexes has never been attempted. Here we explore the polarizable nature of protein–ligand complexes in order to evaluate the importance of this effect. We introduce two indexes describing the polarizability of protein binding sites. These we apply to a large range of pharmaceutically relevant complexes. We offer a recommendation of particular complexes as test systems with which to determine the effects of polarizability and as test cases with which to test the new generation of force fields. Additionally, we provide a tabulation of the amino acid composition of these binding sites and show that composition can be specific for certain classes of proteins. We also show that the relative abundance of some amino acids is different in binding sites than elsewhere in a protein's structure. © 2003 Wiley Periodicals, Inc. Biopolymers 70: 201–211, 2003
Analysis of DNA bending by transient electric birefringence
Authors: Lu Yongjun , Weers Brock D. , Stellwagen Nancy C.
Published: September 2003

Transient electric birefringence has been used to analyze DNA bending in six restriction fragments containing 171, 174, 207, 263, 289, and 471 bp in three different low ionic strength buffers. The target fragments contain sequences corresponding to the apparent bend centers in pUC19 and Litmus 28, previously identified by the circular permutation assay (Strutz, K.; Stellwagen, N. C. Electrophoresis 1996, 17, 989–995). The target fragments migrate anomalously slowly in polyacrylamide gels and exhibit birefringence relaxation times that are shorter than those of restriction fragments of the same size, taken from nonbent regions of the same plasmids. Apparent bend angles ranging from 30° to 41° were calculated for the target fragments by τ‐ratio method. The bend angles of four of the target fragments were independent of temperature from 4°C to 20°C, but decreased when the temperature was increased to 37°C. The bend angles of the other two target fragments were independent of temperature over the entire range examined, 4°–37°C. Hence, the thermal stability of sequence‐dependent bends in random‐sequence DNA is variable. The bend angles of five of the six target fragments were independent of the presence or absence of Mg2+ ions in the solution, indicating most of the target fragments were stably bent or curved, rather than anisometrically flexible. Restriction fragments containing 219 and 224 bp, with sequences somewhat offset from the sequence of the 207 bp fragment, were also studied. Comparison of the τ‐ratios of these overlapping fragments allowed both the bend angle and bend position to be independently determined. These methods should be useful for analyzing sequence‐dependent bending in other random‐sequence DNAs. © 2003 Wiley Periodicals, Inc. Biopolymers 70: 270–288, 2003
Brownian dynamics simulation of helix‐capping motifs
Authors: Shen Tongye , Wong Chung F. , McCammon J. Andrew
Published: September 2003

Helix ‐capping motifs are believed to play an important role in stabilizing α‐helices and defining helix start and stop signals. We performed microsecond scale Brownian dynamics simulations to study ten XAAD sequences, with X = (A,E,I,L,N,Q,S,T,V,Y), to examine their propensity to form helix capping motifs and correlate these results with those obtained from analyzing a structural database of proteins. For the widely studied capping box motif S**D, where the asterisk can be any amino acid residue, the simulations suggested that one of the two hydrogen bonds proposed earlier as a stabilizing factor might not be as important. On the other hand, side‐chain interactions between the capping residue and the third residue downstream on the polypeptide chain might also play a role in stabilizing this motif. These results are consistent with explicit‐solvent molecular dynamics simulations of two capping box motifs found in the proteins BPTI and α‐dendrotoxin. Principal component analysis of the SAAD trajectory showed that the first three principal components, after those corresponding to translational‐rotational motion were removed, accounted for more than half of the conformational fluctuations. The first component separated the conformational space into two parts with the all‐helical conformation and the capping box motif lying largely in one part. The second component, on the other hand, could be used to describe conformational transitions between the all‐helical form and the capping box motif. © 2003 Wiley Periodicals, Inc. Biopolymers 70: 252–259, 2003
Cation effects on swelling of κ‐carrageenan: A photon transmission study
Authors: Kara Selim , Tamerler Candan , Pekcan Önder
Published: September 2003

Swelling behavior of κ‐carrageenan gels in water and KCl solutions was investigated by photon transmission experiments following the preparation of gels in the presence and absence of externally added K+ ion as a gel promoting agent. Transmitted photon intensity, Itr, increased continuously during swelling depending on the carrageenan and ion content in the gel. This increase in Itr was modeled using the Li–Tanaka equation. Both the experimental work and the model showed that the swelling of low carrageenan and ion content gels took less time than that of high ion content gels. It is confirmed that double helices in a swollen gel move much faster in pure water than in KCl solution during swelling processes. Swelling time constants, τ1, and collective diffusion coefficients, Do, were measured for the gels swollen in water and KCl solutions. © 2003 Wiley Periodicals, Inc. Biopolymers 70: 240–251, 2003
MM3 potential energy surfaces of trisaccharides. II. Carrageenan models containing 3,6‐anhydro‐D‐galactose
Authors: Stortz Carlos A. , Cerezo Alberto S.
Published: September 2003

The adiabatic potential energy surfaces (PES) of six trisaccharides—namely 3,6‐An‐α‐D‐Galp‐(1→3)‐β‐D‐Galp‐(1→4)‐3,6‐An‐α‐D‐Galp, β‐D‐Galp‐(1→4)‐3,6‐An‐α‐D‐Galp‐(1→3)‐β‐D‐Galp, and their derivatives sulfated on positions 2 and 4 of the β‐galactose unit—were obtained using the MM3 force field. Each PES was described by a single contour map for which the energy is plotted against the two ψ glycosidic angles, given the small variations of the ϕ glycosidic torsional angle in the low‐energy regions of disaccharide maps. In five of the six examples, the surfaces are those expected from the maps of the disaccharidic repeating units of carrageenans, with less important factors altering the additive effect of both linkages. However, when a sulfate group is present on C2 of a β‐galactose reducing end, a new low‐energy minimum in a different region is produced, originated in a hydrogen bond between the first and third monosaccharidic moieties of the trisaccharide. The flexibility of the β‐linkages is nearly identical to that in their disaccharide counterparts, while that of the α‐linkages is slightly reduced, independent of their presence closer or further away from the reducing end. A fair agreement is observed between the x‐ray fiber diffraction analysis for a κ‐carrageenan double helix and the surfaces obtained for the trisaccharide analogs of that polymer. © 2003 Wiley Periodicals, Inc. Biopolymers 70: 227–239, 2003
Conformational constraints of tyrosine in protein tyrosine kinase substrates: Information about preferred bioactive side‐chain orientation
Authors: Ruzza Paolo , Calderan Andrea , Donella‐Deana Arianna , Biondi Barbara , Cesaro Luca , Osler Alessio , Elardo Stefano , Guiotto Andrea , Pinna Lorenzo A. , Borin Gianfranco
Published: September 2003

The side‐chain orientation of a tyrosine residue located in a peptide, which is an excellent substrate of Syk tyrosine kinase (A. M. Brunati, A. Donella‐Deana, M. Ruzzene, O. Marin, L. A. Pinna, FEBS Letters, 1995, Vol. 367, pp. 149–152), was fixed in the gauche (+) or gauche (−) conformation by using the 7‐hydroxy‐1,2,3,4‐tetrahydro isoquinoline‐3‐carboxylic (Htc) structure. The tyrosine trans conformation was blocked by using an aminobenzazepine‐type (Hba) structure. The proposed side‐chain orientations were confirmed by the analysis of the 1H‐NMR parameters: chemical shifts, coupling constants, and nuclear Overhauser effects to the tyrosine constraints in the different analogs. This “rotamer scan” of the phosphorylatable residue allowed us to generate optimal substrates in terms of both phosphorylation efficiency and selectivity for Syk tyrosine kinase. In contrast, these conformationally restricted tyrosine analogs were not tolerated by the Src‐related tyrosine kinases Lyn and c‐Fgr. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 71: 478–488, 2003
13C CPMAS NMR studies of the elastin‐like polypeptide (LGGVG)n
Authors: Kumashiro Kristin K. , Kurano Tracie L. , Niemczura Walter P. , Martino Marica , Tamburro Antonio M.
Published: September 2003

The elucidation of structure‐function relationships in insoluble elastin is often approached using elastin‐like polypeptides. In this manner, the characterization of the different regions in this extensive biopolymer may be facilitated in a “piece‐wise” manner. Our solid‐state NMR experiments indicate that (LGGVG)n has structural similarities to elastin and some elastin peptides, providing support for the utility of the mimetic peptides. Furthermore, previous NMR and CD studies indicated that the structure of the elastin‐like polypeptide (LGGVG)n in solution is best described as a “conformational ensemble” with a mixture of type I and II β‐turns, in addition to unfolded regions. Our data indicate that the peptide does not adopt a single conformation in the solid state, lending further support to models for elastin that involve significant conformational heterogeneity. © 2003 Wiley Periodicals, Inc. Biopolymers 70: 221–226, 2003
Peptide‐bond modification for metal coordination: Peptides containing two hydroxamate groups
Authors: Ye Yunpeng , Liu Min , Kao Jeff L.‐K. , Marshall Garland R.
Published: September 2003

Peptide‐bond modification via N‐hydroxylation has been explored as a strategy for metal coordination to induce conformational rigidity and orient side chains for specific molecular recognition. N‐Hydroxyamides were prepared by reacting N‐benzyloxyamino acid esters or amides with Fmoc–AA–Cl/AgCN (Fmoc: 9‐fluorenylmethoxycarbonyl; AA: amino acid) in toluene or Fmoc–AA/HATU/DIEA in DMF (HATU: O‐(7‐azabenzotriazol‐lyl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate; DIEA: N,N‐diisopropylethylamine; DMF: N,N‐dimethylformamide), followed by deblocking of benzyl protecting groups. Novel linear and cyclic N,N′‐dihydroxypeptides were efficiently assembled using Fmoc chemistry in solution and/or on a solid support. As screened by electrospray ionization–mass spectroscopy (ESI‐MS), high iron‐binding selectivity and affinity were attainable. Compounds having a spacer of two α‐amino acids between the amino acids bearing the two hydroxamates, i.e., a spacer of 8 atoms, generated 1:1 iron complex species in the gas phase. Moreover, high performance liquid chromatography (HPLC), uv/vis, and 1H‐NMR analyses provided direct evidence for complex formations in solution. Significantly, the representative compound cyclo(Leu–Ψ[CON(OH)]–Phe–Ala–Pro)2 (P8) may serve as a robust metal‐binding scaffold in construction of a metal‐binding library for versatile metal‐mediated molecular recognition. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 71: 489–515, 2003
Study of electrostatic potential surface distribution of wild‐type plastocyanin Synechocystis solution structure determined by homonuclear NMR
Authors: Monleón Daniel , Celda Bernardo
Published: September 2003

Plastocyanin is a small (∼10 kDa), type I blue copper protein that works as an electron donor to photosystem I from cytochrome f in both chloroplast systems and in some strains of cyanobacteria. Comparative studies of the kinetic mechanisms of plastocyanins in different organisms show that the electron transfer from photosystem I happens by simple collision in cyanobacteria but through a intermediate transition complex in green algae and superior plants. Previous work has proved that this effect cannot be explained by structural variations across the different plastocyanins but it can be explained by differences in the electrostatic potential distribution at the protein surface. In that case, minor conformational errors at the amino acid side chain level may imply an important effect in the electrostatic potential distribution calculation. In this work we present a high resolution study of side chain conformation by homonuclear NMR for the reduced wild‐type plastocyanin Synechocystis using intensity ratios for 2D‐NOESY and 2D‐H,H‐TOCSY cross peaks at different mixing times. We also present the corresponding comparison with different plastocyanin structures and the effect in the electrostatic potential distribution at the protein surface. We discuss the importance of indirect J‐coupling information from TOCSY‐type experiments as complement for intraresidue distances derived from NOESY experiments in the determination of side chain orientation and stereo‐specific assignments. © 2003 Wiley Periodicals, Inc. Biopolymers 70: 212–220, 2003
Biosynthesis and biophysical analysis of domains of a yeast G protein‐coupled receptor
Authors: Arevalo Enrique , Estephan Racha , Madeo Jenifer , Arshava Boris , Dumont Mark , Becker Jeffrey M. , Naider Fred
Published: September 2003

The α‐factor receptor(Ste2p) is required for the sexual conjugation of the yeast Saccharomyces cerevisiae. Ste2p belongs to the G protein‐coupled receptor (GPCR) family sharing a common heptahelical transmembrane structure. Biological synthesis of regions of Ste2p fused to a leader protein in Escherichia coli yielded milligram quantities of polypeptides that corresponded to one or two transmembrane domains. Fusion proteins were characterized by polyacrylamide gel electrophoresis, high performance liquid chromatography, and mass spectrometry. CD studies on the fusion proteins in trifluoroethanol:water mixtures indicated the existence of α‐helical structures in the single‐ and the double‐transmembrane domains. NMR experiments were performed in CDCl3:CD3OH:H2O (4:4:1) on the 15N‐labeled single‐transmembrane peptide showing a clear dispersion of the nitrogen–amide proton correlation cross peaks indicative of a high‐purity, uniformly labeled molecule. The results indicate that single‐ and double‐transmembrane domains of a GPCR can be produced by biosynthetic methods in quantities and purity sufficient for biophysical studies. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 71: 516–531, 2003
Solid‐phase synthesis of C‐terminal modified peptides
Authors: Alsina Jordi , Albericio Fernando
Published: September 2003

Solid‐phase synthesis of biomolecules, of which peptides are the principal example, is well established. However, synthetic peptides containing modifications at the carboxy termini are often desired because of their potential therapeutic properties. As a result, there is a necessity for effective solid‐phase strategies for the preparation of peptides with C‐terminal end groups other than the usual carboxylic acid and carboxamide functionalities. The present article primarily reviews literature reports on methods for solid‐phase synthesis of C‐terminal modified peptides. In addition, general information about biological activities and/or synthetic applications of each individual class of peptide is also provided. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 71: 454–477, 2003
ICESEE: A tool for developing engineering courseware
Authors: Ochoa Sergio F. , Pino José A. , Baloian Nelson , Fuller David A.
Published: September 2003

In this article, a computer tool is introduced to support courseware building and evolution in computer science and engineering education. The tool is intended to fulfill requirements, which are typical of this area of education. The tool has been implemented and initially tested. Instructors and students have been satisfied with the tool, therefore, indicating encouraging results. © 2003 Wiley Periodicals, Inc. Comput Appl Eng Educ 11: 53–66, 2003; Published online in Wiley InterScience (www.interscience.wiley.com); DOI 10.1002/cae.10032
Development of web‐based engineering numerical software (WENS) using MATLAB: Applications to linear algebra
Authors: Kim Albert S. , Park Chanho , Park Si‐Hwan
Published: September 2003

In this article, we present Web‐based Engineering Numerical Software (WENS) developed using MATLAB and its Web Server Toolbox (WST) in order to provide basic solution tools in linear algebra, including eigenvalue problems, which are extensively used in engineering applications and education. © 2003 Wiley Periodicals, Inc. Comput Appl Eng Educ 11: 67–74, 2003; Published online in Wiley InterScience (www.interscience.wiley.com); DOI 10.1002/cae.10038
An online thermodynamics courseware
Authors: Ngo C. C. , Lai F. C.
Published: September 2003

This article presents an online Thermodynamics courseware and how it can be used to enhance the learning experience of students. The courseware has been developed to present the materials in a dynamic and interactive fashion. It also includes a review section to help students in the preparation of the Fundamentals of Engineering (FE) Exam. Since its first implementation in summer 2000, this courseware has received overwhelming response from our students. © 2003 Wiley Periodicals, Inc. Comput Appl Eng Educ 11: 75–82, 2003; Published online in Wiley InterScience (www.interscience.wiley.com); DOI 10.1002/cae.10039
A web‐based 2D structural analysis educational software
Authors: Almeida Barretto Saulo Faria , Piazzalunga Renata , Ribeiro Viviane Guimarães
Published: September 2003

This paper describes an educational software developed to be helpful to undergraduate engineering students when learning with structural analysis. The software enables the user to analyze plane‐framed structures, under static or dynamic loading, and it was fully implemented by using web technology. © 2003 Wiley Periodicals, Inc. Comput Appl Eng Educ 11: 83–92, 2003; Published online in Wiley InterScience (www.interscience.wiley.com); DOI 10.1002/cae.10040
Neuro‐Lab: A highly reusable software‐based environment to teach artificial neural networks
Authors: García Roselló Emilio , García Pérez‐Schofield José B. , González Dacosta Jacinto , Pérez‐Cota Manuel
Published: September 2003

It is increasingly usual to find notions of artificial neural networks (ANNs) included in the curricula of many engineering studies. In this article, we present Neuro‐Lab, a simulation software to teach basic concepts of ANNs to undergraduate students of the Computer Engineering School in the University of Vigo (Spain). Neuro‐Lab was designed with the aim of offering a very easy‐to‐use user interface, support of exploratory learning activities, and also high reusability. This feature allows for an easy extension, integration, and interoperation of the software with other applications. © 2003 Wiley Periodicals, Inc. Comput Appl Eng Educ 11: 93–102, 2003; Published online in Wiley InterScience (www.interscience.wiley.com); DOI 10.1002/cae.10042
Flexible Adaptations in the Structure of the tRNA‐Modifying Enzyme tRNA–Guanine Transglycosylase and Their Implications for Substrate Selectivity, Reaction Mechanism and Structure‐Based Drug Design
Authors: Brenk Ruth , Stubbs Milton T. , Heine Andreas , Reuter Klaus , Klebe Gerhard
Published: September 2003

The enzyme tRNA–guanine transglycosylase (TGT, EC 2.4.2.29) catalyses a base‐exchange reaction that leads to anticodon modifications of certain tRNAs. The TGT enzymes of the eubacteria Zymomonas mobilis (Z. mobilis TGT) and Escherichia coli (E. coli TGT) show a different behaviour in the presence of competitive inhibitors. The active sites of both enzymes are identical apart from a single conservative amino acid exchange, namely Tyr106 of Z. mobilis TGT is replaced by a Phe in E. coli TGT. Although Tyr106 is, in contrast to Phe106, hydrogen bonded in the ligand‐free structure, we can show by a mutational study of TGT(Y106F) that this is not the reason for the different responses upon competition. The TGT enzymes of various species differ in their substrate selectivity. Depending on the applied pH conditions and/or induced by ligand binding, a peptide‐bond flip modulates the recognition properties of the substrate binding site, which changes between donor and acceptor functionality. Furthermore interstitial water molecules play an important role in these adaptations of the pocket. The flip of the peptide bond is further stabilised by a glutamate residue that operates as general acid/base. An active‐site aspartate residue, presumed to operate as a nucleophile through covalent bonding during the base‐exchange reaction, shows different conformations depending on the nature of the bound ligand. The induced‐fit adaptations observed in the various TGT complex structures by multiple crystal‐structure analyses are in agreement with the functional properties of the enzyme. In consequence, full understanding of this plasticity can be exploited for drug design.
Structural Investigation of a High‐Affinity MnIIBinding Site in the Hammerhead Ribozyme by EPR Spectroscopy and DFT Calculations. Effects of Neomycin B on Metal‐Ion Binding
Authors: Schiemann Olav , Fritscher Jörg , Kisseleva Natalja , Sigurdsson Snorri Th. , Prisner Thomas F.
Published: September 2003

Electron paramagnetic resonance spectroscopy and density functional theory methods were used to study the structure of a single, high‐affinity MnIIbinding site in the hammerhead ribozyme. This binding site exhibits a dissociation constantKdof 4.4 μMin buffer solutions containing 1 MNaCl, as shown by titrations monitored by continuous wave (cw) EPR. A combination of electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) experiments revealed that the paramagnetic manganese(II) ion in this binding site is coupled to a single nitrogen atom with a quadrupole coupling constantκof 0.7 MHz, an asymmetry parameterηof 0.4, and an isotropic hyperfine coupling constant ofAiso(14N)=2.3 MHz. All three EPR parameters are sensitive to the arrangement of the MnIIligand sphere and can therefore be used to determine the structure of the binding site. A possible location for this binding site may be at the G10.1, A9 site found to be occupied by MnIIin crystals (MacKay et al., Nature1994, 372,68 and Scott et al., Science1996, 274,2065). To determine whether the structure of the binding site is the same in frozen solution, we performed DFT calculations for the EPR parameters, based on the structure of the MnIIsite in the crystal. Computations with the BHPW91 density function in combination with a 9s7p4d basis set for the manganese(II) center and the Iglo‐II basis set for all other atoms yielded values ofκ(14N)=+0.80 MHz,η=0.324, andAiso(14N)=+2.7 MHz, in excellent agreement with the experimentally obtained EPR parameters, which suggests that the binding site found in the crystal and in frozen solution are the same. In addition, we demonstrated by EPR that MnIIis released from this site upon binding of the aminoglycoside antibiotic neomycin B (Kd=1.2 μM) to the hammerhead ribozyme. Neomycin B has previously been shown to inhibit the catalytic activity of this ribozyme (Uhlenbeck et al., Biochemistry1995, 34,11 186).
The Molecular Basis for A‐Site Mutations Conferring Aminoglycoside Resistance: Relationship between Ribosomal Susceptibility and X‐ray Crystal Structures
Authors: Pfister Peter , Hobbie Sven , Vicens Quentin , Böttger Erik C. , Westhof Eric
Published: September 2003

Aminoglycoside antibiotics target the 16S ribosomal RNA (rRNA) bacterial A site and induce misreading of the genetic code. Point mutations of the ribosomal A site may confer resistance to aminoglycoside antibiotics. The influence of bacterial mutations (introduced by site‐directed mutagenesis) on ribosomal drug susceptibility was investigated in vivo by determination of minimal inhibitory concentrations. To determine the origin of the various resistance phenotypes at a molecular level, the in vivo results were compared with the previously published crystal structures of paromomycin, tobramycin, and geneticin bound to oligonucleotides containing the minimal A site. Two regions appear crucial for binding in the A site: the single adenine residue at position 1408 and the non‐Watson–Crick U1406⋅U1495 pair. The effects of mutations at those positions are modulated by the nature of the substituent at position 6′ (either hydroxy or ammonium group) on ring I, by the number of positive charges on the antibiotic, and by the linkage between rings I and III (either 4,5 or 4,6). In particular, the analysis demonstrates: 1) that the C1409–G1491 to A1409–U1491 polymorphism (observed in 15 % of bacteria) is not associated with resistance, which indicates that it does not affect the stacking of ring I on residue 1491, 2) that the high‐level resistance to 6′‐NH${_{3}^{+}}$ aminoglycosides exhibited by the A1408G mutation most probably results from the inability of ring I forming a pseudo base pair with G1408, which prevents its insertion inside the A site helix, and 3) that mutations of the uracil residues forming the U1406⋅U1495 pair either to cytosine or to adenine residues mostly confer low to moderate levels of drug resistance, whereas the U1406C/U1495A double mutation confers high‐level resistance (except for neomycin), which suggests that aminoglycoside binding to the wild‐type A site and its functional consequences strongly depend on a particular geometry of the U1406⋅U1495 pair. The relationships between the resistance phenotypes observed in vivo and the interactions described at the molecular level define the biological importance of the different structural interactions observed by X‐ray crystallography studies.
Evaluation of Bacterial RNase P RNA as a Drug Target
Authors: Willkomm Dagmar K. , Gruegelsiepe Heike , Goudinakis Olga , Kretschmer‐Kazemi Far Rosel , Bald Rolf , Erdmann Volker A. , Hartmann Roland K.
Published: September 2003

RNA has gained increasing importance as a therapeutic target. However, so far mRNAs rather than stable cellular RNAs have been considered in such studies. In bacteria, the tRNA‐processing enzyme RNase P has a catalytic RNA subunit. Fundamental differences in structure and function between bacterial and eukaryotic RNase P, and its indispensability for cell viability make the bacterial enzyme an attractive drug target candidate. Herein we describe two approaches utilized to evaluate whether the catalytic RNA subunit of bacterial RNase P is amenable to inactivation by antisense‐based strategies. In the first approach, we rationally designed RNA hairpin oligonucleotides targeted at the tRNA 3′‐CCA binding site (P15 loop region) of bacterial RNase P RNA by attempting to include principles derived from the natural CopA–CopT antisense system. Substantial inactivation of RNase P RNA was observed for Type A RNase P RNA (such as that in Escherichia coli) but not for Type B (as in Mycoplasma hyopneumoniae). Moreover, only an RNA oligonucleotide (Eco 3′) complementary to the CCA binding site and its 3′ flanking sequences was shown to be an efficient inhibitor. Mutation of Eco 3′ and analysis of other natural RNase P RNAs with sequence deviations in the P15 loop region showed that inhibition is due to interaction of Eco 3′ with this region and occurs in a highly sequence‐specific manner. A DNA version of Eco 3′ was a less potent inhibitor. The potential of Eco 3′ to form an initial kissing complex with the P15 loop did not prove advantageous. In a second approach, we tested a set of oligonucleotides against E. coli RNase P RNA which were designed by algorithms developed for the selection of suitable mRNA targets. This approach identified the P10/11‐J11/12 region of bacterial RNase P RNA as another accessible region. In conclusion, both the P15 loop and P10/11‐J11/12 regions of Type A RNase P RNAs seem to be promising antisense target sites since they are easily accessible and sufficiently interspersed with nonhelical sequence elements, and oligonucleotide binding directly interferes with substrate docking to these two regions.
Stereoselective Synthesis using Immobilized Diels–Alderase Ribozymes
Authors: Schlatterer Jörg C. , Stuhlmann Friedrich , Jäschke Andres
Published: September 2003

Development of artificial ribozymes by in vitro selection has so far, mostly been addressed from the viewpoint of fundamental research. However, such ribozymes also have high potential as selective catalysts in practical syntheses. Immobilization of an active and selective ribozyme is an important step towards this end. A 49‐nucleotide RNA molecule that was previously found to stereoselectively catalyze Diels–Alder reactions between various anthracene dienes and maleimide dienophiles was quantitatively immobilized on an agarose matrix by periodate oxidation of the 3′‐terminal ribose and coupling to a hydrazide moiety. Typical loadings were 45 pmol μL−1 gel. The specific activity was comparable to that of soluble ribozyme, and high enantioselectivities were obtained in catalyzed cycloadditions. The catalytic matrix was found to be stable and could be regenerated about 40 times with only minimal reduction of catalytic activity. Like the soluble ribozyme, the immobilized catalyst stereoselectively converts various diene and dienophile substrates. By using either natural D‐RNA or enantiomeric L‐RNA, both product enantiomers were made synthetically accessible with similar selectivities.
NMR Spectroscopy of RNA
Authors: Fürtig Boris , Richter Christian , Wöhnert Jens , Schwalbe Harald
Published: September 2003

NMR spectroscopy is a powerful tool for studying proteins and nucleic acids in solution. This is illustrated by the fact that nearly half of all current RNA structures were determined by using NMR techniques. Information about the structure, dynamics, and interactions with other RNA molecules, proteins, ions, and small ligands can be obtained for RNA molecules up to 100 nucleotides. This review provides insight into the resonance assignment methods that are the first and crucial step of all NMR studies, into the determination of base‐pair geometry, into the examination of local and global RNA conformation, and into the detection of interaction sites of RNA. Examples of NMR investigations of RNA are given by using several different RNA molecules to illustrate the information content obtainable by NMR spectroscopy and the applicability of NMR techniques to a wide range of biologically interesting RNA molecules.
Oligonucleotide‐Based Knockdown Technologies: Antisense Versus RNA Interference
Authors: Achenbach Tatjana V. , Brunner Bodo , Heermeier Kathrin
Published: September 2003

The postgenomic era is characterized by an almost intimidating amount of information regarding the sequences and expression of previously unknown genes. In response, researchers have developed an increasing interest in functional studies. At the start of such a study, one may have little more than sequence information and bioinformatic annotation. The next step is to hypothesize a potential role in the context of a cell. Testing of the hypothesis needs to be fast, cheap, and applicable to a large number of genes. Knockdown methods that rely on binding of antisense oligonucleotides to mRNA combined with a subsequent functional assay in cell culture fulfil these requirements: sequence information is sufficient for synthesis of active inhibitors. Depending on the in vitro model chosen, knockdown of gene expression can be achieved with medium or even high throughput. The two most popular methods of knockdown in cell culture are the use of antisense oligonucleotides that rely on ribonuclease H (RNAse H)‐dependent cleavage of mRNA, and RNA interference triggered by small double‐stranded RNA molecules. Both methods act in a sequence‐specific manner and can give efficient knockdown. In both cases, researchers struggle with nonspecific “off‐target” effects and the difficulty of site selection. Studies that compare the methods differ in their judgment as to which method is superior.
Cover Picture: Targeting RNA with Small Molecules (ChemBioChem 10/2003)
Authors: Tor Yitzhak
Published: September 2003

The cover picture shows the processes involved in the search for small molecules as potent and selective RNA binders. Motivation comes from the desire to control cell function at the RNA level and to identify novel approaches to specifically combat pathogens by targeting their unique RNA sequences or RNA–protein complexes. Inspiration comes from nature; in particular, from aminoglycosides, a family of naturally occurring antibiotics that has been shown to target the bacterial ribosome. The discovery process involves identifying RNA targets (schematically shown as a ribosome or a virus), devising unique assays (e.g. a solid‐phase assay), and generating the necessary knowledge and lead structures through design, synthesis, and systematic evaluation of biological activity. Further details can be found in the article by Y. Tor on p. 998 ff.
Elective coronary angiography and percutaneous coronary intervention during uninterrupted warfarin therapy
Authors: Jessup David B. , Coletti Andrew T. , Muhlestein Joseph B. , Barry William H. , Shean Frederic C. , Whisenant Brian K.
Published: September 2003

The management of patients anticoagulated with warfarin and referred for coronary angiography presents a substantial challenge to the physician who must minimize risks of periprocedural hemorrhage and thromboembolism. The aim of this study was to evaluate the feasibility and safety of performing diagnostic coronary angiography and percutaneous coronary intervention during uninterrupted warfarin therapy. Patients treated with warfarin were prospectively identified and enrolled in the study. Nineteen diagnostic cardiac catheterizations and six percutaneous coronary interventions were performed in 23 patients. The mean international normalized ratio was 2.4 ± 0.5 (range, 1.8–3.5). Hemostasis was achieved with AngioSeal following 21 procedures and with Perclose following 4 procedures. No patient experienced a predefined endpoint. Specifically, no patient experienced procedure‐related myocardial infarction, major or minor bleeding. We conclude that cardiac catheterization and percutaneous coronary intervention may be considered in the setting of uninterrupted warfarin therapy. Catheter Cardiovasc Interv 2003;60:180–184. © 2003 Wiley‐Liss, Inc.
Catheter‐based 32P β‐radiation after stent implantation in porcine coronary arteries: Role of source‐centering and geographical miss
Authors: Maeng Michael , Busk Martin , Tanderup Kari , Mertz Henrik , Andersen Henning Rud , Thuesen Leif
Published: September 2003

The present study examined the role of source‐centering and geographical miss in vascular brachytherapy. After implantation of 13 mm long stents, 38 coronary arteries in 13 pigs were randomly assigned to centered brachytherapy (n = 13), eccentric brachytherapy (n = 13), or no radiation (n = 12). Geographical miss was avoided by careful placement of a 27 mm 32P β‐radiation source. Restenosis was quantified by angiography, histomorphometry, and intravascular ultrasound at 28 days. Source‐centering led to a significant (P < 0.001) reduction of in‐stent area stenosis (centered radiation, 12% ± 5%; eccentric radiation, 37% ± 21%; control arteries, 41% ± 13%). Despite 7 mm coverage of the edge segments, radiation was found to induce edge stenosis due to neointima formation and constrictive vascular remodeling. We conclude that centered radiation was superior to eccentric radiation in reducing in‐stent luminal narrowing while radiation‐induced edge stenosis was still observed despite extension of the radiation zone to 7 mm beyond the stent edges. Catheter Cardiovasc Interv 2003;60:247–257. © 2003 Wiley–Liss, Inc.
Combined distal embolization protection and rheolytic thrombectomy to facilitate percutaneous revascularization of totally occluded saphenous vein grafts
Authors: Gaitonde Rajdeep S. , Sharma Naveen , von der Lohe Elisabeth , Kalaria Vijay G.
Published: September 2003

Totally occluded saphenous vein grafts are difficult to treat percutaneously with a higher likelihood of distal embolization and slow‐flow or no‐reflow during percutaneous interventions. The PercuSurge system, which utilizes a distal balloon occlusive device, has been shown to improve clinical outcomes during saphenous vein graft (SVG) interventions. This device may not be optimal in the setting of heavy thrombus or debris burden, a situation frequently encountered in totally occluded SVGs. Rheolytic thrombectomy facilitates percutaneous interventions by effectively removing intraluminal thrombus and debris but lacks distal embolization protection. We report our experience with the synergistic use of balloon‐based distal embolization protection (PercuSurge) and rheolytic thrombectomy (AngioJet) to optimize percutaneous revascularization of totally occluded SVGs. Catheter Cardiovasc Interv 2003;60:212–217. © 2003 Wiley‐Liss, Inc.
Transcatheter closure of a ruptured ventricular septum following inferior myocardial infarction and cardiogenic shock
Authors: Schiele Thomas M. , Kozlik‐Feldmann Rainer , Sohn Hae‐Young , Stempfle Hans‐Ulrich , Küchle Claudius , Schopohl Jochen , Theisen Karl , Leibig Marcus , Klauss Volker
Published: September 2003

Elective transcatheter closure of congenital septal defects has emerged as a valuable method, but the clinical experience on occlusion of ventricular septal rupture after myocardial infarction is very limited. We report a case of fatal outcome in a patient with inferior myocardial infarction and cardiogenic shock despite technically successful transcatheter closure of a large complex ventricular septal defect. Catheter Cardiovasc Interv 2003;60:224–228. © 2003 Wiley‐Liss, Inc.
Transcatheter closure of a coronary sinus defect with an Amplatzer septal occluder
Authors: Di Bernardo Stefano , Fasnacht Margrit , Berger Felix
Published: September 2003

We report a successful transcatheter closure of a coronary sinus defect using an Amplatzer septal occluder in a 9.5‐year‐old boy suffering from a significant volume overload due to left‐to‐right shunt. Catheter Cardiovasc Interv 2003;60:287‐290
Endocardial cryotherapy as a novel strategy of improving myocardial perfusion in a patient with severe coronary artery disease
Authors: Segev Amit , Strauss Bradley H. , Coates Geoffry , Freeman Michael R. , Gallo Richard
Published: September 2003

Patients with intractable angina pectoris due to end‐stage coronary artery disease who are not amenable to conventional revascularization provide a therapeutic challenge. We describe the first published case of a young patient with intractable coronary artery disease that was successfully treated by endocardial cryotherapy. Catheter Cardiovasc Interv 2003;60:229–232. © 2003 Wiley‐Liss, Inc.
Modified T‐stenting technique with crushing for bifurcation lesions: Immediate results and 30‐day outcome
Authors: Colombo Antonio , Stankovic Goran , Orlic Dejan , Corvaja Nicola , Liistro Francesco , Airoldi Flavio , Chieffo Alaide , Spanos Vassilis , Montorfano Matteo , Di Mario Carlo
Published: September 2003

We report a new stenting technique employed in 20 consecutive patients to treat true bifurcation lesions using the Cypher stent (Cordis, Warren, NJ). Both stents are advanced at the site of the bifurcation. The proximal marker of the side‐branch stent must be situated in the main branch at a distance of 4–5 mm proximal to the carina of the bifurcation and the main branch stent must cover the bifurcation as well as the protruding segment of the side‐branch stent. The side‐branch stent is deployed first and balloon and wire are removed. The stent deployed in the main branch completely covers and crushes the protruding segment of the side branch stent against the vessel wall of the main branch. Following main‐ and side‐branch predilatation, stents were successfully deployed in all lesions. Final kissing balloon inflation was performed in seven patients. Two patients had in‐hospital myocardial infarction and one patient underwent in‐hospital re‐PTCA due to a dissection distal to a stent. No other major adverse cardiac events were observed in‐hospital and during 1‐month clinical follow‐up. Treatment of bifurcation lesions using crushing stent technique is feasible with acceptable rate of procedural complications. Angiographic follow‐up is necessary to prove the advantage of this specific technique to give complete coverage of the ostium of the side branch with a drug‐eluting stent. Catheter Cardiovasc Interv 2003;60:145–151. © 2003 Wiley‐Liss, Inc.
Neonatal transcatheter occlusion of a large coronary artery fistula with Amplatzer duct occluder
Authors: Khan Mohammad D. , Qureshi Shakeel A. , Rosenthal Eric , Sharland Gurleen K.
Published: September 2003

A large fistula from the right coronary artery to the right atrium was diagnosed in a fetus at 22 weeks of gestation. Following delivery, the baby developed cardiac failure. The fistula was occluded at 3 weeks of age by transcatheter implantation of an Amplatzer duct occluder. Large coronary fistulas can be closed safely with a device even at this early age. Vigilance for ischemic events is required following implantation because of the rare possibility of thrombus extension proximally into the native coronary artery or spasm of the coronary artery. Catheter Cardiovasc Interv 2003;60:282–286. © 2003 Wiley‐Liss, Inc.
Large pseudoaneurysm after left main trunk stenting sealed by polytetrafluorethylene‐covered stent
Authors: Nameki Mizuo , Ishiwata Sugao , Momomura Shin‐ichi
Published: September 2003

Left main trunk (LMT) aneurysm is very rare and the management remains uncertain. We describe a patient who developed a pseudoaneurysm from coronary perforation during stent implantation in LMT and was then treated with polytetrafluorethylene (PTFE)‐covered stent graft. PTFE‐covered stent is considered to be a valid strategy for LMT aneurysms. Catheter Cardiovasc Interv 2003;60:233–235. © 2003 Wiley‐Liss, Inc.
Percutaneous closure of a giant coronary arteriovenous fistula using multiple devices in a 12‐day‐old neonate
Authors: Holzer Ralf , Waller B. Rush , Kahana Madelyn , Hijazi Ziyad M.
Published: September 2003

Coronary arteriovenous fistulas (CAVFs) are uncommon in children and are rarely reported in neonates. Larger fistulas usually require either surgical closure or percutaneous closure using a variety of different devices. Device closure of large fistulas in the immediate neonatal period has not been reported. We report the first case of a gigantic CAVF in a 12‐day‐old neonate (2.4 kg) where successful device closure was achieved in a staged approach using a 12 mm Amplatzer muscular VSD device, seven flipper coils, a 10/8 mm Amplatzer duct occluder device, as well as a 9 mm Gianturco Grifka vascular occlusion device. No complications were encountered and the patient was subsequently weaned off the ventilator. Catheter Cardiovasc Interv 2003;60:291–294. © 2003 Wiley‐Liss, Inc.
Bare delivery of medium and large stents in patients with congenital heart disease
Authors: Vance Michael S.
Published: September 2003

The medium and large stents favored by pediatric cardiologists are typically delivered through a long sheath placed distal to the target lesion. However, long sheath placement can be problematic. The aim of the study was to determine whether stents can be safely delivered without using a long sheath in patients with congenital heart disease. Between July 2001 and October 2002, primary bare stent delivery was attempted in 17 consecutive patients with 21 target lesions. All 21 stents were successfully delivered without employing a long sheath. The series demonstrates that medium and large stents can be safely delivered without a long sheath in patients with congenital heart disease. Furthermore, eliminating the long sheath can improve stent delivery and allow new options when addressing bifurcation lesions. Catheter Cardiovasc Interv 2003;60:276–281. © 2003 Wiley‐Liss, Inc.