Central Loop Research Articles

Genetic Analysis of the Structure and Function of 7SK Small Nuclear Ribonucleoprotein (snRNP) in Cells

The positive transcription elongation factor b (P-TEFb), comprised of cyclin-dependent kinase 9 (CDK9) and cyclins T1 (CycT1) or T2 (CycT2), activates eukaryotic transcription elongation. In growing cells, P-TEFb exists in active and inactive forms. In the latter, it is incorporated into the 7SK small nuclear ribonucleoprotein, which contains hexamethylene bisacetamide-induced proteins (HEXIM) 1 or 2, La-related protein 7 (LaRP7), methyl phosphate capping enzyme, and 7SK small nuclear RNA (7SK). HEXIM1 inhibits the kinase activity of CDK9 via interactions between 7SK, HEXIM1, and CycT1. LaRP7 and methyl phosphate capping enzyme interact with 7SK independently of HEXIM1 and P-TEFb. To analyze genetic interactions between HEXIM1 and/or LaRP7 and 7SK using a cell-based system, we established artificial heterologous RNA tethering assays in which reporter gene expression depended on interactions between selected regions of 7SK and its cognate binding partners fused to a strong activator. This system enabled us to map the HEXIM1- and LaRP7- binding regions of 7SK. Assays with various mutant 7SK plasmid targets revealed that the 5'U-Ubulge and central loop of stem-loop I or RNA motif 3 of 7SK are required for transactivation, suggesting that HEXIM1 and CycT1 form a combinatorial binding surface for 7SK. Moreover, a region in HEXIM1 C-terminal to its previously mapped RNA-binding motif was also required for interactions between HEXIM1 and 7SK. Finally, a tyrosine-to-alanine mutation in HEXIM1, which is critical for its inhibitory effect on CDK9, changed HEXIM1 into an activator. These cell-based assays elucidate this important aspect of transcription elongation in vivo.

Molecular evolution of a novel family of putative calcium transporters.

The UPF0016 family is a group of uncharacterized membrane proteins, well conserved through evolution and defined by the presence of one or two copies of an E-Φ-G-D-(KR)-(ST) consensus motif. Our previous results have shown that two members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and might form a new group of cation/Ca2+ exchangers. Most members of the family are made of two homologous clusters of three transmembrane spans, separated by a central loop and assembled with an opposite orientation in the membrane. However, some bacterial members of the family have only one cluster of transmembrane domains. Among these ‘single-domain membrane proteins’ some cyanobacterial members were found as pairs of adjacent genes within the genome, but each gene was slightly different. We performed a bioinformatic analysis to propose the molecular evolution of the UPF0016 family and the emergence of the antiparallel topology. Our hypotheses were confirmed experimentally using functional complementation in yeast. This suggests an important and conserved function for UPF0016 proteins in a fundamental cellular process. We also show that members of the UPF0016 family share striking similarities, but no primary sequence homology, with members of the cation/Ca2+ exchangers (CaCA) superfamily. Such similarities could be an example of convergent evolution, supporting the previous hypothesis that members of the UPF0016 family are cation/Ca2+ exchangers.

Discussion on Method of Eliminating Large Loop Source TEM Border Effect

Large loop source TEM method has played an important role in minerals and engineering exploration. But there is a strong border effect when one explains by central loop later-time apparent resistivity. In this paper, a method was used to calculate all-time apparent resistivity by magnetic dipoles composing loop. For 1D forward data, all-time apparent resistivity was computed and compared with later-time apparent resistivity. Conclusions have been drawn that all-time apparent resistivity has no border effect on homogeneous ground model; but for complex layered model, all-time apparent resistivity border effect is reflected in vicinity of false extreme point of apparent resistivity curve, and is much weaker than later-time apparent resistivity border effect. The conclusions are conducive to a correct understanding of using all-time apparent resistivity to eliminate border effect and guide correct interpretation of measurement data.

The folding of 5'-UTR human G-quadruplexes possessing a long central loop.

G-quadruplexes are widespread four-stranded structures that are adopted by G-rich regions of both DNA and RNA and are involved in essential biological processes such as mRNA translation. They are formed by the stacking of two or more G-quartets that are linked together by three loops. Although the maximal loop length is usually fixed to 7 nt in most G-quadruplex-predicting software, it has already been demonstrated that artificial DNA G-quadruplexes containing two distal loops that are limited to 1 nt each and a central loop up to 30 nt long are likely to form in vitro. This report demonstrates that such structures possessing a long central loop are actually found in the 5'-UTRs of human mRNAs. Firstly, 1453 potential G-quadruplex-forming sequences (PG4s) were identified through a bioinformatic survey that searched for sequences respecting the requirement for two 1-nt long distal loops and a long central loop of 2-90 nt in length. Secondly, in vitro in-line probing experiments confirmed and characterized the folding of eight candidates possessing central loops of 10-70 nt long. Finally, the biological effect of several G-quadruplexes with a long central loop on mRNA expression was studied in cellulo using a luciferase gene reporter assay. Clearly, the actual definition of G-quadruplex-forming sequences is too conservative and must be expanded to include the long central loop. This greatly expands the number of expected PG4s in the transcriptome. Consideration of these new candidates might aid in elucidating the potentially important biological implications of the G-quadruplex structure.

Paralogous radiations of PIN proteins with multiple origins of noncanonical PIN structure.

The plant hormone auxin is a conserved regulator of development which has been implicated in the generation of morphological novelty. PIN-FORMED1 (PIN) auxin efflux carriers are central to auxin function by regulating its distribution. PIN family members have divergent structures and cellular localizations, but the origin and evolutionary significance of this variation is unresolved. To characterize PIN family evolution, we have undertaken phylogenetic and structural analyses with a massive increase in taxon sampling over previous studies. Our phylogeny shows that following the divergence of the bryophyte and lycophyte lineages, two deep duplication events gave rise to three distinct lineages of PIN proteins in euphyllophytes. Subsequent independent radiations within each of these lineages were taxonomically asymmetric, giving rise to at least 21 clades of PIN proteins, of which 15 are revealed here for the first time. Although most PIN protein clades share a conserved canonical structure with a modular central loop domain, a small number of noncanonical clades dispersed across the phylogeny have highly divergent protein structure. We propose that PIN proteins underwent sub- and neofunctionalization with substantial modification to protein structure throughout plant evolution. Our results have important implications for plant evolution as they suggest that structurally divergent PIN proteins that arose in paralogous radiations contributed to the convergent evolution of organ systems in different land plant lineages.

Short and long sympathetic-sensory feedback loops in white fat.

We previously demonstrated white adipose tissue (WAT) innervation using the established WAT retrograde sympathetic nervous system (SNS)-specific transneuronal viral tract tracer pseudorabies virus (PRV152) and showed its role in the control of lipolysis. Conversely, we demonstrated WAT sensory innervation using the established anterograde sensory system (SS)-specific transneuronal viral tracer, the H129 strain of herpes simplex virus-1, with sensory nerves showing responsiveness with increases in WAT SNS drive. Several brain areas were part of the SNS outflow to and SS inflow from WAT between these studies suggesting SNS-SS feedback loops. Therefore, we injected both PRV152 and H129 into inguinal WAT (IWAT) of Siberian hamsters. Animals were perfused on days 5 and 6 postinoculation after H129 and PRV152 injections, respectively, and brains, spinal cords, sympathetic, and dorsal root ganglia (DRG) were processed for immunohistochemical detection of each virus across the neuroaxis. The presence of H129+PRV152-colocalized neurons (~50%) in the spinal segments innervating IWAT suggested short SNS-SS loops with significant coinfections (>60%) in discrete brain regions, signifying long SNS-SS loops. Notably, the most highly populated sites with the double-infected neurons were the medial part of medial preoptic nucleus, medial preoptic area, hypothalamic paraventricular nucleus, lateral hypothalamus, periaqueductal gray, oral part of the pontine reticular nucleus, and the nucleus of the solitary tract. Collectively, these results strongly indicate the neuroanatomical reality of the central SNS-SS feedback loops with short loops in the spinal cord and long loops in the brain, both likely involved in the control of lipolysis or other WAT pad-specific functions.

Research on the Application of a 3-m Transmitter Loop for TEM Surveys in Mountainous Areas

Large loop transient electromagnetic (TEM) is a widely used surface exploration method for mapping electrically conductive bodies. However, when working in the mountainous areas of China, it is difficult to lay out large enough transmitter loops for conventional surveys. An option for this type of terrain is a newly designed TEM survey configuration that uses a small 3-m square loop in combination with a high current (1,000 A to 2,000 A) TEM transmitter to excite a powerful EM field for detecting targets at greater depths. The detection depth of the system is analyzed. After comparing the responses as well as the detection depths between this special system and standard large loop configurations, it is shown that: 1) a small loop system can result in a detection depth similar to that of a large loop system; and 2) a small loop system is superior to a large loop during shallow depth target detection as well as in mountainous areas. Sufficient signal intensity can be obtained by a small central loop configuration equipped with a high current transmitter and a large equivalent area receiver coil. A large trial survey was conducted on the surface of mountainous areas in the Shanxi Province of China. This TEM survey showed that the 3-m by 3-m transmitter loop system was an effective method for deep TEM sounding in mountainous areas.

How HIV-1 Nef hijacks the AP-2 clathrin adaptor to downregulate CD4

The Nef protein of HIV-1 downregulates the cell surface co-receptor CD4 by hijacking the clathrin adaptor complex AP-2. The structural basis for the hijacking of AP-2 by Nef is revealed by a 2.9 Å crystal structure of Nef bound to the α and σ2 subunits of AP-2. Nef binds to AP-2 via its central loop (residues 149-179) and its core. The determinants for Nef binding include residues that directly contact AP-2 and others that stabilize the binding-competent conformation of the central loop. Residues involved in both direct and indirect interactions are required for the binding of Nef to AP-2 and for downregulation of CD4. These results lead to a model for the docking of the full AP-2 tetramer to membranes as bound to Nef, such that the cytosolic tail of CD4 is situated to interact with its binding site on Nef. DOI: http://dx.doi.org/10.7554/eLife.01754.001.

Chapter 9 - The effects of head-up and head-down tilt on central respiratory chemoreflex loop gain tested by hyperoxic rebreathing

Central respiratory chemosensitivity is mediated via chemoreceptor neurons located throughout brain stem tissue. These receptors detect proximal CO2/[H(+)] (i.e., controller gain) and modulate breathing in a classic negative feedback loop. Loop gain (responsiveness) is the theoretical product of controller (chemoreceptors), mixing/feedback (cardiovascular and cerebrovascular systems), and plant (pulmonary system) gains. The level of chemoreceptor stimulation is determined by interactions between mixing and plant gains. The extent to which steady-state changes in body position may affect central chemoreflex loop gain in response to CO2 is unclear. Because of the potential effects of tilt on pulmonary mechanics, we hypothesized that plant gain would be altered by head-up and head-down tilt (HUT, HDT) during hyperoxic rebreathing, which theoretically isolates plant gain by eliminating systemic arterial-tissue gradients. Sixteen subjects (eight females) underwent hyperoxic rebreathing tests on a tilt table to quantify central chemoreflex loop gain in five steady-state positions: 90° HUT, 45° HUT, supine, 45° HDT, and 90° HDT. Respiratory responses (tidal volume, VT; frequency, fR; minute ventilation, VE) were quantified during steady-state and increases in CO2 during rebreathing by linear regression above the ventilatory recruitment threshold (VRT). Using one-factor analysis of variance, we found that there were no differences in the respiratory responses between the five positions (VRT, P=0.711; VT, P=0.290; fR, P=0.748; VE, P=0.325). Our findings suggest that during steady-state orthostatic stress, the ability of subjects to mount a normal ventilatory response to increased CO2 was unaffected, despite any potential changes in pulmonary mechanics associated with positional challenges.

The M3 phosphorylation motif has been functionally conserved for intracellular trafficking of long-looped PIN-FORMEDs in the Arabidopsis root hair cell

BackgroundPIN-FORMED (PIN) efflux carriers contribute to polar auxin transport and plant development by exhibiting dynamic and diverse asymmetrical localization patterns in the plasma membrane (PM). Phosphorylation of the central hydrophilic loop (HL) of PINs has been implicated in the regulation of PIN trafficking. Recently, we reported that a phosphorylatable motif (M3) in the PIN3-HL is necessary for the polarity, intracellular trafficking, and biological functions of PIN3. In this study, using the root hair system for PIN activity assay, we investigated whether this motif has been functionally conserved among long-HL PINs.ResultsRoot hair-specific overexpression of wild-type PIN1, 2, or 7 greatly inhibited root hair growth by depleting auxin levels in the root hair cell, whereas overexpression of M3 phosphorylation-defective PIN mutants failed to inhibit root hair growth. Consistent with this root hair phenotype, the PM localization of M3 phosphorylation-defective PIN1 and PIN7 was partially disrupted, resulting in less auxin efflux and restoration of root hair growth. Partial formation of brefeldin A-compartments in these phosphorylation-mutant PIN lines also suggested that their PM targeting was partially disrupted. On the other hand, compared with the PIN1 and PIN7 mutant proteins, M3-phosphorylation-defective PIN2 proteins were almost undetectable. However, the mutant PIN2 protein levels were restored by wortmannin treatment almost to the wild-type PIN2 level, indicating that the M3 motif of PIN2, unlike that of other PINs, is implicated in PIN2 trafficking to the vacuolar lytic pathway.ConclusionsThese results suggest that the M3 phosphorylation motif has been functionally conserved to modulate the intracellular trafficking of long-HL PINs, but its specific function in trafficking has diverged among PIN members.

Three-Dimensional Nonlinear Force-Free Field Reconstruction of Solar Active Region 11158 by Direct Boundary Integral Equation

A 3-D coronal magnetic field is reconstructed for NOAA 11158 on Feb 14, 2011. A GPU-accelerated direct boundary integral equation (DBIE) method is implemented. This is about 1000 times faster than the original DBIE used on solar NLFFF modeling. Using the SDO/HMI vector magnetogram as the bottom boundary condition, the reconstructed magnetic field lines are compared with the projected EUV loop structures from different views three-dimensionally by SDO/AIA and STEREO A/B spacecraft simultaneously for the first time. They show very good agreement so that the topological configurations of the magnetic fields can be analyzed, thus its role in the flare process of the active region can be better understood. A quantitative comparison with some stereoscopically reconstructed coronal loops shows that the present averaged misalignment angles are at the same order as the state-of-the-art results obtained with reconstructed coronal loops as prescribed conditions and better than other NLFFF methods. It is found that the observed coronal loop structures can be grouped into bundles of closed and open loops with some central bright coronal loops around the polarity inversion line (PIL). The reconstructed highly-shearing magnetic field lines agree very well with the low-lying S-shaped filament channel along PIL. They are in a pivot position to all other surrounding coronal structures, and a group of electric current lines co-aligned with the central bright EUV loops overlying the filament channel is also obtained. This central lower-lying magnetic field loop system must have played a key role in powering the flare. It should be noted that while a strand-like coronal feature along PIL may be related to the filament, one cannot simply attribute all the coronal bright features along PIL to manifestation of the filament without any stereoscopically information. It shows that DBIE is rigorous and effective.

Response and Test of Whole Space Transient Electromagnetic Field in Mine Goaf Water

The transient electromagnetic method (TEM) has sensitive characteristics of low resistivity of water containing. The equivalent plane method based on whole space theory is used in coal mine goaf water TEM detecting. The mine goaf water was forward simulation. The geoelectric model of mine goaf water was equivalented to low resistivity layer thickness. The geoelectric model was forward calculated, and it was differential imaged. Then it found the apparent longitudinal conductance differential imaging reflect the most sensitive. The field experiment was conducted in two mine goaf water in Xiezhuang coal mine. The central loop device was selected as working device. The launch wire frame is length of 2m and 8 turns. A receiving device is receiving probe and the effective area is 212m2. The Water distribution range of goaf water in-300 of the seven mining area in front of retaining wall is large, and the water body is deep.

Kinetic model of double-stranded RNA formation during long RNA replication by Qβ replicase

Qβ replicase is an RNA-dependent RNA polymerase, which synthesizes the complementary RNA using a single-stranded RNA as a template. The formation of non-replicable double-stranded RNA (dsRNA) by hybridization between newly synthesized RNA and the template RNA hinders the broader application of Qβ replicase. Here, we developed a kinetic model of Qβ RNA replication consisting of two reaction pathways of dsRNA formation, which quantitatively explains the dynamics of dsRNA formation of three template RNAs. We also found that part of the Qβ phage genomic RNA sequence including the central hairpin loop significantly decreases the rate of dsRNA formation.

Squeezing at Entrance of Proton Transport Pathway in Proton-translocating Pyrophosphatase upon Substrate Binding

Homodimeric proton-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) is indispensable for many organisms in maintaining organellar pH homeostasis. This unique proton pump couples the hydrolysis of PPi to proton translocation across the membrane. H(+)-PPase consists of 14-16 relatively hydrophobic transmembrane domains presumably for proton translocation and hydrophilic loops primarily embedding a catalytic site. Several highly conserved polar residues located at or near the entrance of the transport pathway in H(+)-PPase are essential for proton pumping activity. In this investigation single molecule FRET was employed to dissect the action at the pathway entrance in homodimeric Clostridium tetani H(+)-PPase upon ligand binding. The presence of the substrate analog, imidodiphosphate mediated two sites at the pathway entrance moving toward each other. Moreover, single molecule FRET analyses after the mutation at the first proton-carrying residue (Arg-169) demonstrated that conformational changes at the entrance are conceivably essential for the initial step of H(+)-PPase proton translocation. A working model is accordingly proposed to illustrate the squeeze at the entrance of the transport pathway in H(+)-PPase upon substrate binding.

PMIIND-an MPI-based population density simulation framework

MIIND [1] is the first publicly available implementation of population density algorithms. Like neural mass models, they model at the population level, rather than that of individual neurons, but unlike neural mass models, they consider the full neuronal state space. The central concept is a population density, a probability distribution function that represents the probability of a neuron being in a certain part of state space. Neurons will move through state space by their own intrinsic dynamics or driven by synaptic input. When individual spikes do not matter but only population averaged quantities are considered, these methods outperform direct simulations using neuron point models by a factor 10 or more, whilst (at the population level) producing identical results to simulations of spiking neurons. This is in general not true for neural mass models. Population density methods also relate closely to analytic evaluations of population dynamics. The evolution of the density function is given by a partial differential equation (PDE). In [3] a generic method was presented for solving this equation efficiently, both for small synaptic efficacies (diffusion limit; the PDE becomes a Fokker-Planck equation) and for large ones (finite jumps). We demonstrated that for leaky-integrate-and-fire (LIF) neurons this method reproduces analytic results [1] and uses of the order of 0.2 s to model 1s simulation time of infinitely large population of spiking LIF neurons. We now have developed this method to apply to any 1D neuron point model [3], not just LIF neurons and demonstrated the technique on quadratic-integrate-and-fire neurons. We are therefore in the position to model large heterogeneous networks of spiking neurons very efficiently. A potential bottleneck is MIIND's serial simulation loop. We developed an MPI implementation of MIIND's central simulation loop starting from a fresh code base, and addressed serialization, which is now done at the level of individual cores. Central assumption in the set up is that firing rates are communicated, not individual spikes, so bandwidth requirements are low. Latency is potentially a problem, but with the use of latency hiding techniques good scalability for up to 64 cores has been achieved ondedicated clusters. The scalability was verified with a simple model of cortical waves in a hexagonal network of populations with balanced excitation-inhibition. pMIIND is available on Sourceforge, through its git repository: git://http://miind.sourceforge.net A CMake-based install procedure is provided. Since pMIIND is set up as a C++ framework, it is possible to define one's own algorithms and still take advantage of the MPI-based simulation loop.

G-Quadruplex conformational change driven by pH variation with potential application as a nanoswitch

BackgroundG-Quadruplex is a highly polymorphic structure, and its behavior in acidic condition has not been well studied. MethodsCircular dichroism (CD) spectra were used to study the conformational change of G-quadruplex. The thermal stabilities of the G-quadruplex were measured with CD melting. Interconversion kinetics profiles were investigated by using CD kinetics. The fluorescence of the inserted 2-Aminopurine (Ap) was monitored during pH change and acrylamide quenching, indicating the status of the loop. Proton NMR was adopted to help illustrate the change of the conformation. ResultsG-Quadruplex of specific loop was found to be able to transform upon pH variation. The transformation was resulted from the loop rearrangement. After screening of a library of diverse G-quadruplex, a sequence exhibiting the best transformation property was found. A pH-driven nanoswitch with three gears was obtained based on this transition cycle. ConclusionsCertain G-quadruplex was found to go through conformational change at low pH. Loop was the decisive factor controlling the interconversion upon pH variation. G-Quadruplex with TT central loop could be converted in a much milder condition than the one with TTA loop. It can be used to design pH-driven nanodevices such as a nanoswitch. General significanceThese results provide more insights into G-quadruplex polymorphism, and also contribute to the design of DNA-based nanomachines and logic gates.

Synthesis, characterization and in vitro activity of thrombin-binding DNA aptamers with triazole internucleotide linkages

A series of DNA aptamers bearing triazole internucleotide linkages that bind to thrombin was synthesized. The novel aptamers are structurally analogous to the well-known thrombin-inhibiting G-quadruplexes TBA15 and TBA31. The secondary structure stability, binding affinity for thrombin and anticoagulant effects of the triazole-modified aptamers were measured. A modification in the central loop of the aptamer quadruplex resulted in increased nuclease resistance and an inhibition efficiency similar to that of TBA15. The likely aptamer-thrombin binding mode was determined by molecular dynamics simulations. Due to their relatively high activity and the increased resistance to nuclease digestion imparted by the triazole internucleotide linkages, the novel aptamers are a promising alternative to known DNA-based anticoagulant agents.

SAT0413 Sympathetic Skin Response in Diabetic Patients with Soft Tissue Rheumatism of the Hand

BackgroundMusculoskeletal complications of the hand may be very debilitating for the diabetic patient. The most commonly recognized complications are trigger finger, tenosynovitis, Dupuytren’s contrcture, carpal tunnel syndrome (CTS), and limited...

The design of a new truncated and engineered alpha1-antitrypsin based on theoretical studies: an antiprotease therapeutics for pulmonary diseases

Alpha 1- antitrypsin (α1AT) a 54 kDa glycoprotein is a protease inhibitor. In the absence of α1AT, elastase released by lung macrophages, was not inhibited and lead to elastin breakdown and pulmonary problems such as emphysema or COPD. α1AT has three site of N-glycosylation and a characteristic reactive central loop (RCL). As small-scale medicines are preferred for pulmonary drug delivery, in this study α1ATs (1, 2, 3, 4 and 5) were engineered and shortened from the N-terminal region. In order to investigate the effect of different mutations and the deletion of 46 amino acids theoretical studies were performed. Homology modeling was performed to generate the 3D structure of α1ATs. The 10 ns Molecular Dynamic (MD) simulations were carried out to refine the models. Results from MD and protein docking showed that α1AT2 has the highest binding affinity for neutrophil elastase, provided the basis for the experimental phase in which sequences from the five α1AT constructs were inserted into the expression vector pGAPZα and expressed in the yeast Pichia pastoris. Although, the α1AT2 construct has the highest inhibitory activity even more that the native construct (α1AT5), results indicated the presence of protease inhibitory function of all the proteins' construct against elastase.

Linezolid Resistance in Brazilian Staphylococcus hominis Strains Is Associated with L3 and 23S rRNA Ribosomal Mutations

Among different clinical staphylococcal strains, linezolid resistance has been mainly mediated by mutations in the central loop of domain V of 23S rRNA, with the G2576T mutation being the most prevalent.…