High-definition Structure Research Articles

Impact of E163R cTnT Mutation on Cardiac Mechanics and Energetics in a Murine Model

Introduction: Many of cTnT mutations linked to cardiomyopathies fall the TNT1 domain/N terminal tail region of unresolved high definition structure. This region (∼94-170) of cTnT is critical to Tm binding and contraction regulation. Here, the impact of the E163R mutation in cTnT-TNT1 on contractile function and tension cost was investigated using intact and skinned preparations from WT and transgenic mouse hearts.Methods: Left and right ventricular trabeculae were dissected from non-transgenic wild type (WT) and heterozygous (Δ160E or E163R) mouse hearts and mounted isometrically to record twitch tension or, when skinned, Ca2+ activated force. Myofibrillar ATPase activity was measured by fluorimetric enzyme coupled assay (de Tombe and Stienen, 1995).Results: Myocardium of E163R mice shows: (i) no change of myosin isoform expression (ii) maintained peak isomentric twitch tension at all stimulation frequencies, (iii) prolonged time to peak and time to 50% relaxation, with preserved rate-adaptation of twitch duration, (iv) changes of the short-term interval force relationship and increased occurrence of spontaneous contractions. No significant differences were found in maximum Ca2+ activated tension of E163R and WT skinned trabeculae. However, Ca2+ sensitivity of tension was significantly increased in E163R skinned trabeculae when compared with WT. As to the economy of force maintenance, preliminary experiments suggest an increase of tension cost in trabeculae from E163R hearts. Resting ATPase activity also tended to be higher in E163R preparations. Kinetics of force development and relaxation will be assessed on single myofibrils, isolated from the same hearts.Conclusions: Both primary sarcomeric changes and secondary E-C coupling alterations contribute to mechanical impairment in E163R cTnT mutant myocardium. Supported by: EC Grant n. 241577 (BIG-Heart)

Formation of (3+1) G-Quadruplexes with a Long Loop by Human Telomeric DNA Spanning Five or More Repeats

Structural studies of human telomeric repeats represent an active field of research with potential applications toward the development of specific telomeric quadruplex-targeting drugs for anticancer treatment. To date, high-definition structures were limited to DNA sequences containing up to four GGGTTA repeats. Here we investigate the formation of G-quadruplexes in sequences spanning five to seven human telomeric repeats using NMR, UV, and CD spectroscopy. A (3+1) G-quadruplex with a long propeller loop was isolated from a five-repeat sequence utilizing a guanine-to-inosine substitution. A simple approach of selective site-specific labeling of guanine residues was devised to rigorously determine the folding topology of the oligonucleotide. The same scaffold could be extrapolated to six- and seven-repeat sequences. Our results suggest that long human telomeric sequences consisting of five or more GGGTTA repeats could adopt (3+1) G-quadruplex structures harboring one or more repeat(s) within a single loop. We report on the formation of a Watson-Crick duplex within the long propeller loop upon addition of the complementary strand, demonstrating that the long loop could serve as a new recognition motif.

Determination of a High-Definition Structure of Antimicrobial Piscidin-3 At the Water-Bilayer Interface

Piscidins are a family of naturally occurring host-defense antibiotics that are short, cationic, and amphipathic in structure. Extensive NMR studies of membrane-bound Piscidin 1 (p1), a 22-mer, have shown that the peptide is composed of two alpha-helical segments that lie in the plane of the lipid bilayer. These segments are joined by a kink at residue glycine 13 (G13). Previous studies of Piscidin 3 (p3), another isoform of piscidin, have revealed decreased antimicrobial and hemolytic activity when compared to p1.

Novel Classes of Dimer Antitumour Drug Candidates

Polyvalency in the biological world is defined as the simultaneous binding of multiple ligands to one receptor. Polyvalency can increase the affinity of the polyvalent ligand by 100-1000 fold over the monovalent ligand. Such phenomenon has been employed to design polyvalent toxin inhibitors. Bivalency is a similar approach where two ligands are joined together with a linker to form a homo- or hetero-dimer with an increase in affinity by up to several hundred fold over the monovalent ligand. This review will summarize the recent advancement in designing bivalent inhibitors to be used as antitumour agents. Some dimers (e.g. artemisinin homo-dimer) simply increase the affinity of the monovalent ligands without detailed knowledge of the target. Other dimers are designed with well-characterized targets, for example, jesterone dimer (inhibiting Rel/NF-kappaB) and 3,3'-diindolymethane and their derivatives (inhibiting Akt and NFkappaB). Some dimers are designed based on the high definition structure between ligand and target (e.g. benzodiazepine and daunorubicin interacting with DNA). Heterodimers have also been produced by combining either two different antitumor drugs (e.g. cis-platin/acridine or cis-platin/naphthalimide) or combining one antitumor candidate (artemisinin) with a molecule which can increase the efficacy of the former (transferrin receptor). Finally we will discuss the design of bivalent inhibitors of the P-glycoprotein (ABCB1; MDR or P-gp) to overcome the problem of antitumor resistance.

Issues in development of NIR micro spectrometer system for on-line process monitoring of milk product

The development of a near-infrared (NIR) spectrometer system for on line monitoring of fat in milk processing is outlined. This system uses microsystems optical components fabricated using the LIGA technique. This technique is used in microelectronics to fabricate high definition structures in silicon. For production of large numbers of components significant cost reductions can be realised over conventional methods. To date these LIGA fabricated spectrometers have found applications in colour analysis and in quality analysis in diamonds, but have not been integrated into on-line process applications. This work outlines initial installation and test of such a system in a milk processing environment. The possibility of low cost optical systems being introduced for process control may occur with future work. This technology, operating in the near infra red (800–1100 nm), enables a number of applications to be investigated. The system hardware is outlined and initial tests with the first field installation are discussed. The system has been shown to be sensitive to required changes in fat content.

An intramolecular i-motif: the solution structure and base-pair opening kinetics of d(5mCCT3CCT3ACCT3CC)

We present a high-definition structure of d(5mCCT3CCT3ACCT3CC), a DNA sequence which resembles a four-times repeat of the C-rich strand of telomeres and centromeres. The structure is monomeric. The CC stretches form four hemi-protonated C·C base-pairs, belonging to two parallel-stranded duplexes which intercalate head-to-tail into an i-motif core. The four grooves of the core are similar to those observed previously in i-motif tetramers, with P-P distances around 0.9 nm and 1.4 nm for the narrow and wide grooves, respectively. At 0°C, the structure is formed even at pH 7, despite the required protonation of cytidine pairs, suggesting that it may be biologically relevant.The intercalation topology of the i-motif core is read off the pattern of inter-residue cross-peaks along each groove: between H1′ protons across the narrow grooves, and between amino and H2′ protons across the wide grooves. In the hemi-protonated C·C pairs, the imino proton is shared equally between the two bases, as shown by the equal intensities of the NOESY cross-peaks between the imino proton and the two cis amino protons of the pair. Short inter-sugar distances and the direction of CH1′ bonds are consistent with CH1′⋯O4′ hydrogen bonds across the narrow grooves, as suggested by Berger et al. (1996). Proc. Natl. Acad. Sci. USA, 93, 12116–12121.At one extremity of the i-motif core, the T3A linker loops across one of the two wide grooves. It extends the core by stacking of A11, which also forms a strongly propeller-twisted reverse-Hoogsteen pair with T8. At the other extremity, the two T3linkers loop side by side across the two narrow grooves, extending the core by stacking of a T5·T16 pair which connects the two linkers. In this T·T pair between parallel strands, the hydrogen bonds are from imino proton to O4, and the base-pair lifetime is 6 ms at 0°C. The structures of segments 1 to 7 and 12 to 18, which form the i-motif core and the T3loops, are related by a 2-fold pseudo-symmetry: the geometries and environment are so similar that the NOESY spectra are barely resolved.These various interactions illustrate how linker sequences may affect the stability, intercalation topology and folding pattern of the intramolecular i-motif.

Model of a Quinary Structure between Krebs TCA Cycle Enzymes: A Model for the Metabolon

The enzymes which are responsible for catalyzing sequential reactions in several metabolic pathways have been proposed to be highly organized in supramolecular complexes termed metabolons. However, the in situ existence of these weak complexes is difficult to demonstrate because many of them are dissociated during isolation due to dilution effects. Consequently, the metabolon concept is subject to controversy. A model system consisting of genetically prepared bienzymatic fusion proteins has been used to immobilize sequential metabolic enzymes in close proximity and to demonstrate possible kinetic advantages of metabolons. These experiments use the sequential Krebs TCA cycle enzymes from yeast mitochondrial malate dehydrogenase (MDH), citrate synthase (CS), and aconitase (ACO). Using the porcine high-definition structures of these three enzymes, we have performed computer-modeling studies in order to understand how the molecules may interact. Among the thousands of docking orientations we have tried, one was found to respond to the structural and experimental constraints from the results obtained with the yeast fusion proteins. Interestingly, this quinary structure model shows substantial interacting surface areas with spatial and electrostatic complementarities which make the complex thermodynamically stable. This structure also contains an unbroken electrostatically favorable channel connecting the active sites of ACO and CS, as well as the one previously reported between CS and MDH active sites. Charged amino acids which could be involved in interactions stabilizing the complex have been identified. This model will be used as the basis for further experimental work on the structure of the Krebs TCA cycle metabolon.

Solution structure and base pair opening kinetics of the i-motif dimer of d(5mCCTTTACC): a noncanonical structure with possible roles in chromosome stability

Repetitive cytosine-rich DNA sequences have been identified in telomeres and centromeres of eukaryotic chromosomes. These sequences play a role in maintaining chromosome stability during replication and may be involved in chromosome pairing during meiosis. The C-rich repeats can fold into an 'i-motif' structure, in which two parallel-stranded duplexes with hemiprotonated C.C+ pairs are intercalated. Previous NMR studies of naturally occurring repeats have produced poor NMR spectra. This led us to investigate oligonucleotides, based on natural sequences, to produce higher quality spectra and thus provide further information as to the structure and possible biological function of the i-motif. NMR spectroscopy has shown that d(5mCCTTTACC) forms an i-motif dimer of symmetry-related and intercalated folded strands. The high-definition structure is computed on the basis of the build-up rates of 29 intraresidue and 35 interresidue nuclear Overhauser effect (NOE) connectivities. The i-motif core includes intercalated interstrand C.C+ pairs stacked in the order 2*.8/1.7*/1*.7/2.8* (where one strand is distinguished by an asterisk and the numbers relate to the base positions within the repeat). The TTTA sequences form two loops which span the two wide grooves on opposite sides of the i-motif core; the i-motif core is extended at both ends by the stacking of A6 onto C2.C8+. The lifetimes of pairs C2.C8+ and 5mC1.C7+ are 1 ms and 1 s, respectively, at 15 degrees C. Anomalous exchange properties of the T3 imino proton indicate hydrogen bonding to A6 N7 via a water bridge. The d(5mCCTTTTCC) deoxyoligonucleotide, in which position 6 is occupied by a thymidine instead of an adenine, also forms a symmetric i-motif dimer. However, in this structure the two TTTT loops are located on the same side of the i-motif core and the C.C+ pairs are formed by equivalent cytidines stacked in the order 8*.8/1.1*/7*.7/2.2*. Oligodeoxynucleotides containing two C-rich repeats can fold and dimerize into an i-motif. The change of folding topology resulting from the substitution of a single nucleoside emphasizes the influence of the loop residues on the i-motif structure formed by two folded strands.

Etching of AC thin film electroluminescent devices

Realization of high definition ac thin film electroluminescent (ACTFEL) devices require fine control of geometric definition. Suitable etching processes need to be defined to address the requirements of both isotropic and anisotropic etching profiles, with good etching rates and high selectivity to underlying Si features. Three different etching processes are analyzed and compared with reference to these parameters. Wet chemical processing provides high etch rates and high selectivities for the ACTFEL materials. Using phosphoric acid leads to an infinite selectivity to all ACTFEL materials. Unfortunately, wet chemical etching has poor control for step profiles. Ion beam etching and sputter etching, however can produce both isotropic and anisotropic patterns, but are comparatively slow and suffer from poor selectivity to the Si based materials. Although individually no method is ideal, for full ZnS:Mn ACTFEL device fabrication these results show that a combination of two of these etching processes will provide the necessary geometry's for very high definition structures.

Structuring of bubble overlays by reactive sputter etching in an Ar–H2O atmosphere

The structuring of magnetic bubble propagation overlay patterns by sputter etching, using photoresist as a mask, becomes imperfect for high definition structures. In the past, reactive sputter etching in an O2 containing atmosphere has been introduced as an alternative, the mask material being a metal such as V, Ti, or Cu. The present paper describes how the etch rate ratio between NiFe and Ti can be considerably increased to at least 10 by replacing oxygen with water vapor. The masking Ti layer can than be extremely thin, which has a beneficial effect on the patterning accuracy.