Hairpin Configuration Research Articles

Helical hairpin structure of influenza hemagglutinin fusion peptide stabilized by charge-dipole interactions between the N-terminal amino group and the second helix.

The fusion domain of the influenza coat protein hemagglutinin HA2, bound to dodecyl phosphocholine micelles, was recently shown to adopt a structure consisting of two antiparallel α-helices, packed in an exceptionally tight hairpin configuration. Four interhelical H(α) to C═O aliphatic H-bonds were identified as factors stabilizing this fold. Here, we report evidence for an additional stabilizing force: a strong charge-dipole interaction between the N-terminal Gly(1) amino group and the dipole moment of helix 2. pH titration of the amino-terminal (15)N resonance, using a methylene-TROSY-based 3D NMR experiment, and observation of Gly(1 13)C' show a strongly elevated pK = 8.8, considerably higher than expected for an N-terminal amino group in a lipophilic environment. Chemical shifts of three C-terminal carbonyl carbons of helix 2 titrate with the protonation state of Gly(1)-N, indicative of a close proximity between the N-terminal amino group and the axis of helix 2, providing an optimal charge-dipole stabilization of the antiparallel hairpin fold. pK values of the side-chain carboxylate groups of Glu(11) and Asp(19) are higher by about 1 and 0.5 unit, respectively, than commonly seen for solvent-exposed side chains in water-soluble proteins, indicative of dielectric constants of ε = ∼30 (Glu(11)) and ∼60 (Asp(19)), placing these groups in the headgroup region of the phospholipid micelle.

DNA Unwinding Mechanism of a RecQ Helicase from Arabidopsis Thaliana Investigated with Magnetic Tweezers

Helicases are ATP hydrolysis driven molecular motors that processively unwind double stranded DNA. In this study we investigate RecQ2 helicase from Arabidopsis thaliana (AtRecQ2) which plays an important role in genomic maintenance. We use high resolution magnetic tweezers in order to probe the unwinding of a DNA hairpin by this enzyme in real time under different external forces. We find an unwinding rate of 7-9 bp/s which is slow compared to many prokaryotic helicases. Applied forces between 5 and 12 pN only weakly affect this parameter, while the AT versus GC content of the unwound DNA has a significant impact. The weak force- but the relatively strong sequence dependence of DNA unwinding is in disagreement with a passive ratchet unwinding mechanism. High-resolution measurements reveal that AtRecQ2 unwinds the DNA in 3-4 bp steps. Beyond the behavior of AtRecQ2 during unwinding we analyze its behavior on single-strand DNA. While failing to detect single-strand DNA translocation, the data in contrary suggests that AtRecQ2 diffuses on single stranded DNA. Such a weak contact to single-strand DNA is supported by the observation that even on a stretched hairpin configuration the enzyme is capable of repetitive shuffling, i.e. the instantaneous restart of DNA unwinding after a terminated event. Based on this we hypothesize that AtRecQ2 switches between a processive unwinding-mode on double-stranded DNA and a diffusive-mode on single-stranded DNA in order to keep a target hairpin constantly open or to search for a new distant target fork.

Test results and analyses of conductor short samples for China first PF conductor

The first China PF conductor sample (CNPF1) which was made of one single cable section with hairpin configuration and without bottom joint was fabricated with Chinese NbTi strands and assembled at ENEA and CEA according to the requirements of the SULTAN test facility. The sample was equipped with temperature sensors and voltage taps at CEA according to the test program. The test program included DC performance, cyclic loading, AC loss, and MQE test. The sample exhibited a good performance which fit well with the requirement in the procurement arrangement (PA). But most of the current sharing temperature ( T cs ) tests showed a suddenly voltage take-off or fast voltage transition with take-off electric field below the threshold of 10 μV/m. The temperature could be considered as quench temperature ( T q ) but not exactly T cs . At 35 kA and background field of 3.5 T, the temperature T q was 6.94 K. Even after 2000 cycles at the condition of 6.5 T and 19 kA, the T q remained unchanged.

Electrogenerated chemiluminescence detection of adenosine based on triplex DNA biosensor

A novel electrogenerated chemiluminescence (ECL) biosensor based on the construction of triplex DNA for the detection of adenosine was designed. The ECL biosensor employs an aptamer as a molecular recognition element, and quenches ECL of tris(2,2′-bipyridine) ruthenium (Ru(bpy) 3 2+) by ferrocenemonocarboxylic acid (FcA). Through self-assembly technology, the ECL probe of thiolated hairpin adenosine aptamer tagged was self-assembled onto the surface of a gold electrode with an ECL signal producer Ru(bpy) 3 2+ derivative (Ru-DNA-1). The adenosine aptamer, including a section of triplex characteristic chain, formatted triplex DNA with two other DNAs (DNA-2, Fc-DNA-3) in the presence of triplex DNA binder coralyne chloride (CORA). Fc-DNA-3 was tagged with an ECL quencher ferrocenemonocarboxylic acid (FcA), a quenching probe. In the presence of adenosine, the aptamer sequence (Ru-DNA-1) prefers to form the aptamer–adenosine complex with hairpin configuration and the switch of the DNA-1 occurs in conjunction with the generation of a strong ECL signal owing to the dissociation of a quenching probe. Meanwhile, a control experiment was performed; the ECL-duplex biosensor was designed to detect adenosine. The detection limits were 2.7 × 10 −10 mol L −1 and 2.3 × 10 −9 mol L −1 for the ECL-triplex DNA biosensor and ECL-duplex DNA biosensor, respectively, which demonstrated that the ECL-triplex DNA biosensor improved the sensitivity and specificity greatly.

Comparison of Gadobenate Dimeglumine and Gadodiamide in the Evaluation of Spinal Vascular Anatomy with MR Angiography

Spinal MRA has been increasingly used to evaluate non-invasively the spinal cord vasculature. Our aim was to prospectively compare gadobenate dimeglumine with gadodiamide in the assessment of the normal spinal cord vasculature by using contrast-enhanced MRA, with the hypothesis that high T1 relaxivity gadolinium compounds may improve visualization of the intradural vessels. Twenty subjects underwent 2 temporally separated contrast-enhanced spinal MRAs with gadobenate dimeglumine and gadodiamide (0.2 mmol/kg). Two blinded observers rated postprocessed images on the following qualitative parameters: background homogeneity, sharpness, vascular continuity, and contrast enhancement. Delineation of the ASA, AKA, hairpin configuration of the ASA-AKA connection, and visualized ASA length were recorded. Each observer indicated which of the 2 matched studies he or she thought was of the best overall diagnostic quality. According to both observers gadobenate dimeglumine was superior to gadodiamide in the representation of vascular continuity and contrast (P value < .05). Background homogeneity was not significantly different between the studies. One observer favored gadobenate dimeglumine over gadodiamide in the demonstration of vascular sharpness, while the second observer did not find any significant difference between contrast agents. There was no significant difference between contrast agents in the visualization of the ASA, AKA, hairpin-shaped ASA-AKA connection, and visualized length of the ASA. The overall quality of the gadobenate dimeglumine-enhanced MRA was deemed superior in 15 and 16 cases, respectively, by the 2 observers. Improved image quality and vascular contrast enhancement of spinal MRA at 1.5T is achieved with high T1 relaxivity gadolinium contrast agents compared with conventional agents at equivalent doses.

Silencing effect study of Akt2-siRNA aimed at lung cancer cell NCI-H446.

Lung cancer is one of the most frequent malignant tumors and at the first place of mortality in today's world, It becomes more and more clear that the initiation and development of lung cancer is multistep processes involved in many genes. RNAi gets more and more attention in gene therapy because of its high efficiency and specificity. With RNAi technology, eukaryotic expression vectors aimed at AKT2 siRNA was designed and constructed, which is transfected into human small lung cancer cell line NCI-H446. The silencing effect was observed. Using the known sequence of AKT2's mRNA (M95936), we designed two oligonucleotides that have short hairpin configuration and one oligonucleotide as control. DNA double strands was annealed, and cloned into vectorpGEM-T. Then, blue-white selection and T7/SP6 PCR was used to screen the positive clones, the target DNA and the express vector pRNAT-U6.2 were cut down by BamHI and XhoI restriction enzyme, and linked with each other; use PCR test to filtrate the positive clones and sequenced the target DNA, then transfected it into human small lung cancer cell line (NCI-H446), cultured and screened by G418, RT-PCR was used to detect the expression level of AKT2 mRNA in NCI-H446. validate the positive sequence by PCR and, transfect them to lung cancer cell line (NCI-H446), AKT2-mRNA's expression was evidently declined. The expression vector pRNAT-U6.2-AKT2 was constructed successfully and the expression of Akt2 mRNA in small lung cancer cell line (NCI-H446) was obviously silenced after transfection.

A Biochemically Defined System for Coding Joint Formation in V(D)J Recombination

V(D)J recombination is one of the most complex DNA transactions in biology. The RAG complex makes double-stranded breaks adjacent to signal sequences and creates hairpin coding ends. Here, we find that the kinase activity of the Artemis:DNA-PKcs complex can be activated by hairpin DNA ends in cis, thereby allowing the hairpins to be nicked and then to undergo processing and joining by nonhomologous DNA end joining. Based on these insights, we have reconstituted many aspects of the antigen receptor diversification of V(D)J recombination by using 13 highly purified polypeptides, thereby permitting variable domain exon assembly by using this fully defined system in accord with the 12/23 rule for this process. The features of the recombination sites created by this system include all of the features observed in vivo (nucleolytic resection, P nucleotides, and N nucleotide addition), indicating that most, if not all, of the end modification enzymes have been identified.

Experimental Results of Large-Current Capacity HTS Conductors

Large-current capacity high-temperature superconducting (HTS) conductors suitable for high-field large-scale DC magnets, such as of helical-type fusion reactor FFHR, are being designed by employing YBCO tapes. As the first step of R&D of such an HTS conductor, we have fabricated short conductor samples using Bi-2223/Ag tapes. The HTS conductor was made by simply stacking and soldering Bi-2223/Ag tapes inside a copper sheath. The HTS conductor sample was fabricated in a hair-pin configuration and was tested under a bias magnetic field of 8 T. The conductor was thermally insulated by epoxy and GFRP and was conduction-cooled by liquid helium from the ends. Resistive heaters attached to the surface of the GFRP-insulated conductor were used to increase the temperature of the conductor for testing at elevated temperatures up to 30 K. The critical currents of the HTS conductor at 4.2 K, 10 K, 20 K, and 30 K were measured in a bias magnetic field of 8 T. The stability margin of the HTS conductor was examined in conduction-cooled conditions at different temperatures. The conductor was found to be very stable even at high currents closer to the critical currents.

Optimal contrast medium injection protocols for the depiction of the Adamkiewicz artery using 64-detector CT angiography

To determine the optimal contrast medium injection protocol for demonstrating the Adamkiewicz artery (AKA) using 64-detector CT angiography (CTA). CTA was performed using 64-detector CT. The study population consisted of 80 patients (mean age 67.2 years) with aortoiliac diseases. In the first 60 patients 540 mg I/kg body weight was administered over 25s. The patients were randomly assigned to three protocols with imaging started at 15 (protocol A-1), 18 (A-2), or 21s (A-3) after triggering (threshold 150 HU). The other 20 received 720 mg I/kg body weight with an imaging delay of 18s (protocol B). Two radiologists evaluated the presence of the AKA and measured the attenuation of the aorta and AKA. Aortic enhancement was 360.4, 348, 279.3, and 372 HU for protocols A-1, A-2, A-3, and B, respectively. There was no significant difference between the A-1 and A-2 protocols (Tukey-Kramer test, p=0.73); however, aortic enhancement was significantly lower in A-3 than A-1 and A-2 (p<0.01). There was no significant difference between A-2 and B (p=0.40). AKA attenuation was 69.3, 91.9, 94.6, and 105.4 HU for protocols A-1, A-2, A-3, and B, respectively. There was no significant difference between the A-2 and A-3 protocols (p=0.91); however, AKA attenuation was significantly lower with A-1 than A-2 or A-3 (p=0.01). AKA attenuation was significantly lower with A-2 than B (p=0.03) and there was a significant difference between A-2 (50%) and B (95%) in the depiction of the hairpin configuration of the AKA (p=0.02). For the demonstration of the AKA at CTA, the optimal protocol used an imaging delay of 18s after triggering and an iodine dose of 720 mg I/kg body weight.

Multiscale Modeling of Dislocation Mechanisms in Nanoscale Multilayered Composites

AbstractIt is well known that the mechanical behavior of nanoscale multilayered composites is strongly governed by single dislocation mechanisms and dislocation-interface interactions. Such interactions are complex and multiscale in nature. In this work, two such significant effects are modeled within the dislocation dynamics-continuum plasticity framework: elastic properties mismatch (Koehler image forces) and interface shearing in the case of weak interfaces. The superposition principle is used to introduce the stress fields due to both effects solved for by finite elements. The validation of both methodologies is presented. Furthermore, it was found that the layer-confined threading stress of a dislocation in hair-pin configuration increases if the layer is surrounded by layers made of a stiffer material and that this strengthening effect grows more significant as the layer thickness decreases. The observation made through molecular dynamics, that weak interfaces act as dislocation sinks, was also captured with our approach. A dislocation is attracted to the interface independent of its sign or character. Also the force increases sharply as the dislocation approaches the interface. These findings agree with published molecular dynamics simulations and dislocation-based equilibrium models of this type of interaction.

Role of the ϵ Subunit of Thermophilic F1-ATPase as a Sensor for ATP

The epsilon subunit of F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) has been shown to bind ATP. The precise nature of the regulatory role of ATP binding to the epsilon subunit remains to be determined. To address this question, 11 mutants of the epsilon subunit were prepared, in which one of the basic or acidic residues was substituted with alanine. ATP binding to these mutants was tested by gel-filtration chromatography. Among them, four mutants that showed no ATP binding were selected and reconstituted with the alpha(3)beta(3)gamma complex of TF(1). The ATPase activity of the resulting alpha(3)beta(3)gammaepsilon complexes was measured, and the extent of inhibition by the mutant epsilon subunits was compared in each case. With one exception, weaker binding of ATP correlated with greater inhibition of ATPase activity. These results clearly indicate that ATP binding to the epsilon subunit plays a regulatory role and that ATP binding may stabilize the ATPase-active form of TF(1) by fixing the epsilon subunit into the folded conformation.

Immobilized molecular beacons: A new strategy using UV-activated poly(methyl methacrylate) surfaces to provide large fluorescence sensitivities for reporting on molecular association events

We have designed appropriately prepared solid supports consisting of poly(methyl methacrylate) (PMMA) that provide enhanced performance levels for molecular beacons (MBs) that are used for recognizing and reporting on signature DNA sequences in solution. The attachment of primary amine-containing MBs to the PMMA surface was carried out by UV activating the PMMA to produce surface-confined carboxylate groups, which could then be readily coupled to the MBs using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) chemistry. The fluorescence properties of the MBs covalently attached onto this UV-activated PMMA surface were evaluated and compared with the same MBs immobilized onto glass supports. We observed improved limits of detection for the solution complement to the MBs when immobilized onto PMMA, and this was attributed to both the lower autofluorescence levels exhibited by PMMA at the detection wavelengths used and the improved quenching efficiency of the MBs when in their closed hairpin configuration when strapped to a PMMA surface as opposed to glass. As an example of the utility of the PMMA-based immobilization strategies developed for MBs, we report on the analysis of complementary DNAs specific for fruitless ( fru) and Ods-site homeobox ( OdsH) genes extracted from Drosophila melanogaster fruit flies. The fru gene functions in the central nervous system, where it is necessary for sex determination and male courtship behavior, whereas the OdsH gene is involved in the regulation of transcription.

A single stem-loop structure in Tacaribe arenavirus intergenic region is essential for transcription termination but is not required for a correct initiation of transcription and replication

The genome of Tacaribe virus (TV), prototype of the New World arenaviruses, comprises two RNA segments each encoding two proteins in an ambisense orientation separated by an intergenic region (IGR). We used a TV minireplicon system to investigate the nature of the IGR structures required for transcription termination. We show that efficient generation of subgenomic (SG) RNAs is related to a single hairpin structure comprising a stem with variable numbers of uninterrupted base pairs and stabilized by high Δ G values. The low ability of highly stable hairpin structures comprising bulged stems to support SG RNA synthesis suggested the importance of hairpin configuration for transcription termination. Neither the sequences downstream nor those upstream from the hairpin played a role in SG RNA accumulation. We also show that independently of the IGR structure the unencapsidated mRNAs contained short stretches of nontemplated bases at their 5′ ends which are capped, whereas the 5′ ends of the nucleocapsid-associated antiminigenomes contained an uncapped extra residue. The results support the conclusions that: (i) transcription termination in TV is related to a structural element that is independent of sequence and (ii) the transcription termination signal is not required for a correct initiation of transcription and replication.

An autonomous fueled machine that replicates catalytic nucleic acid templates for the amplified optical analysis of DNA

Here we describe a protocol for the amplified detection of a target DNA using a DNA/FokI-based replicating cutting machine. The protocol is based on the design of a sensing hairpin oligonucleotide that is opened upon hybridization with the analyte DNA. The endonuclease FokI binds to the double-stranded complex and cleaves it to a "cutter" unit. The "cutter" unit reacts with a fuel oligonucleotide to generate and amplify the signal. The fuel molecule is an oligonucleotide in a hairpin configuration with a fluorophore/quencher pair attached to the 5' and 3' ends. Formation of the duplex between the cutter and the fuel leads to the scission of the duplex by FokI, leading to a second, replicated "cutter", a fluorescent waste product, and to the regeneration of the original "cutter" unit. The autonomous replication of the "cutter" unit, as a result of the primary recognition of the analyte DNA, leads to the amplified fluorescent detection of the analyte DNA with a sensitivity limit of 1 x 10(-14) M. The operation of the machine and the sensing process are monitored by the fluorescence generated by the waste product. Here we apply the protocol, which takes about 2 h to complete, to analyze a Tay-Sachs genetic disorder mutant DNA.

SIMS studies of polymeric tertiary structures in monolayers: Polysiloxane helical coil structures

A static SIMS study was performed using poly(dimethyl siloxane) (PDMS) as a model system to investigate the effects of tertiary structure on the ion formation mechanism and ion formation probability of fragments in the high mass region (>1000 Da). PDMS produced from anionic polymerization with a narrow polydispersity, can form well-ordered helically coiled monolayers using Langmuir–Blodgett methods at the air water interface, either in a hairpin configuration, if the endgroups are functional or as flat helically coiled chains. Reflection absorption Fourier transform infrared (RA-FTIR) spectroscopic analysis shows the changes in bonding between flat helically coiled PDMS and cast films from dilute solvents, which produces a random coil configuration. Differences between the model systems in infrared spectrum show evidence of changes in structure, producing variation in band formation. Also observed are changes in the band shape and relative peak area between the model systems. These polymeric changes translate into differences in the relative intensities of fragments formed in the repeating pattern of clusters in the high mass ToF-SIMS spectra of the ordered versus the random cast films. Both statistical chain break analysis and molecular mechanics simulations of structure are used to support the analysis.

Pyrene-labeled deoxyuridine and deoxyadenosine: fluorescent discriminating phenomena in their duplex and hairpin oligonucleotides

Pyrene-labeled deoxyuracil and deoxyadenine units are useful unnatural nucleobases. These fluorescent nucleobase analogues allow strong interstrand stacking interactions to compensate for a loss of hydrogen bonding and exhibit a range of different emission intensities when they form duplexes with one another. These findings may provide new insights into the design of new probes and nucleobase analogues for applications in molecular biology. For this purpose, we have prepared an hairpin molecular beacon (MB) that incorporates an excimer unit in its closed state, and have utilized lambda(max) changing to discriminate between match and mismatch. This hairpin configuration is attractive because the synthesis of such an MB is relatively simple and inexpensive because it does not require two distinct processes to prepare the fluorophore and quencher.

A low cost joint for the ITER PF coils, design and test results

The Poloidal Field (PF) coils of the International Thermonuclear Experimental Reactor (ITER) are designed using of NbTi cable-in-conduit conductors carrying a current up to 45 kA. Each PF coil consists of several modules wound in double pancakes. Joints connect that double pancakes electrically in series at the coil periphery. In order to minimize the manufacturing cost maintaining a high reliability, a low cost joint design was developed at CRPP and tested in SULTAN test facility in the frame of ITER task for PF coils. The concept of the joint is a single fully welded stainless steel box and one pitch joint through sectioned copper saddle blocks. The joint sample includes two full-scale joints, one in the hairpin configuration at the bottom of the sample and one as an overlap joint at mid length of the sample. Lifting the sample in the SULTAN facility, either the hairpin or the overlap joint can be placed in the center of the facility and tested under ITER-relevant operating conditions. The low cost joint design for the ITER PF coils is described and the test results (DC and AC loss) are presented in this paper.

Possible active origin of replication in the double stranded extended form of the left terminus of LuIII and its implication on the replication model of the parvovirus

BackgroundThe palindromic termini of parvoviruses have proven to play an essential role as origins of replication at different stages during the replication of their viral genome. Sequences from the left-end telomere of MVM form a functional origin on one side of the dimer replicative form intermediate. In contrast, the right-end origin can operate in its closed replicative form hairpin configuration or as a fully duplex linear sequence derived from either arm of a palindromic tetramer intermediate. To study the possibility that the LuIII left hairpin has a function in replication, comparable to that described for MVM, the replication of a minigenome containing two copies of the LuIII left terminus (LuIII Lt-Lt) was studied. ResultsThe data presented demonstrates that LuIII Lt-Lt was capable of replicating when NS1 helper functions were provided in trans. This extended hairpin, capable of acting as an origin of replication, lacks the arrangement of the specific domains present in the dimer duplex intermediate of MVM, the only active form of the left hairpin described for this parvovirus.ConclusionsThese findings suggest that the left hairpin of LuIII has an active NS1 driven origin of replication at this terminus in the double stranded extended form. This difference between LuIII and MVM has great implications on the replication of these viruses. The presence of origins of replication at both the left and right termini in their natural hairpin form can explain the unique encapsidation pattern observed for LuIII hinting on the mechanism used by this virus for the replication of its viral genome.

Hairpin conformation of amphidinols possibly accounting for potent membrane permeabilizing activities

Amphidinols are a unique dinoflagellate metabolite with potent antifungal activity. We examined membrane permeabilizing action by amphidinol analogues with structural variations in polyhydroxy and polyene moieties. Consequently, the polyene and polyhydroxy moieties turned out to play important roles in binding to lipid bilayer membrane and in forming ion-permeable pore/lesion across membrane, respectively. NMR-constrained modeling experiments have revealed for the fist time that amphidinols in membrane generally take a hairpin configuration, which plausibly accounts for their potent antifungal and other membrane permeabilizing activities.

Flexible magnetic filaments as micromechanical sensors.

We propose a new micromechanical approach to probe bending rigidity at molecular scale. Long flexible filaments made of magnetic colloids and linkers are shown to adopt under magnetic field a hairpin configuration. Measuring the hairpin curvature as a function of the field intensity and the linker length from diffracted light allows us to deduce the linker bending rigidity kappa. The technique is presented for two types of linkers: a spontaneously adsorbing polymer and a grafted biomolecular.