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At least 109 records · Page 6Linked to original sources

Atrial flutter vector loops derived from the surface ECG: does the plane of the loop correspond anatomically to the macroreentrant circuit?

We hypothesized that if the right atrial circuit during isthmus dependent atrial flutter provides the dominant contribution to the surface electrocardiogram (ECG), the three-dimensional vector loop of the flutter waves would primarily be in a plane approximately parallel to the tricuspid ring. Twenty vectorcardiograms of isthmus dependent atrial flutter derived from 12-lead ECGs of 19 patients recorded prior to radiofrequency ablation were analyzed. The plane of each loop, described by azimuth and elevation angles relative to the frontal plane, was estimated with two methods: 1) plane of maximum loop area and 2) plane of best fit. The plane of maximum loop of the loops had mean azimuth of -58 +/- 37 degrees. and elevation of 15 +/- 15 degrees. The plane of best fit of the loops had mean azimuth of -50 +/- 46 degrees and elevation of 15 +/- 14 degrees. Thus, clinical implications include the potential to predict atrial flutter mechanisms prior to intracardiac mapping.

Atrial Flutter↗

Apical loop-internal loop interactions: a new RNA-RNA recognition motif identified through in vitro selection against RNA hairpins of the hepatitis C virus mRNA.

We performed in vitro selection of oligoribonucleotides in order to identify high-affinity motifs recognizing RNA hairpins located at the 3' end (SL1) and at the 5' end (domain IV of the internal ribosome entry site) of the hepatitis C virus mRNA. We selected aptamers constituted by an internal loop complementary to the SL1 apical loop, flanked by G-C-rich double-stranded regions, able to form complexes with a K(d) of 70 nM, at 37 degrees C under ionic conditions close to intracellular ones. The complex involves selective apical loop (SL1)-internal loop (aptamer) interactions. Similar structurally organized aptamers were independently identified against domain IV and were shown to also give rise to such complexes. Apical loop-internal loop interaction could constitute a new recognition motif allowing specific intra- or intermolecular RNA-RNA association.

Base Sequence↗

NMR structures of loop B RNAs from the stem-loop IV domain of the enterovirus internal ribosome entry site: a single C to U substitution drastically changes the shape and flexibility of RNA.

The 5'-untranslated region of positive-strand RNA viruses harbors many cis-acting RNA structural elements that are important for various viral processes such as replication, translation, and packaging of new virions. Among these is loop B RNA of the stem-loop IV domain within the internal ribosomal entry site (IRES) of enteroviruses, including Poliovirus type 1 (PV1). Studies on PV1 have shown that specific recognition of loop B by the first KH (hnRNP K homology) domain of cellular poly(rC)-binding protein 2 (PCBP2) is essential for efficient translation of the viral mRNA. Here we report the NMR solution structures of two representative sequence variants of enteroviral loop B RNA. The two RNA variants differ at only one position (C vs U) within a six-nucleotide asymmetric internal loop sequence that is the binding site for the PCBP2 KH1 domain. Surprisingly, the two RNAs are drastically different in the overall shape and local dynamics of the bulge region. The RNA with the 5'-AUCCCU bulge sequence adopts an overall L shape. Its bulge nucleotides, especially the last four, are highly flexible and not very well defined by NMR. The RNA with the 5'-AUUCCU bulge sequence adopts an overall U shape, and its bulge sequence exhibits only limited flexibility. A detailed analysis of the two RNA structures and their dynamic properties, as well as available sequence data and known KH domain-RNA complex structures, not only provides insights into how loop B RNA might be recognized by the PCBP2 KH1 domain but also suggests a possible correlation between structural flexibility and pre-existing structural features for protein recognition.

5' Untranslated Regions↗

NCp7 activates HIV-1Lai RNA dimerization by converting a transient loop-loop complex into a stable dimer.

Nucleocapsid protein 7 (NCp7), the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, was shown to strongly potentiate the dimerization of the retroviral genomic RNA. This process involves the interaction of two retroviral RNA monomer subunits near their 5'-ends. A region located upstream from the splice donor site was recently identified as being responsible for the formation of dimeric HIV-1 RNA. This region appeared to be confined within a stem-loop structure, with an autocomplementary sequence in the loop. In an in vitro study of spontaneous dimer formation, we reported that the 77-402 RNA transcript forms two distinct dimers differing in their thermostability: D37 and D55. We identified D37 as a "kissing" complex structure, formed via a loop-loop interaction between the two monomers, and D55 as a double stranded structure involving all nucleotides of the stem-loop via canonical base pairing. In this report, we have characterized the role of NCp7 in the HIV-1Lai RNA dimerization process by using in vitro dimerization assays with RNA transcripts of different lengths and dimer thermal dissociation. Our results show that the nucleocapsid protein NCp7 activates RNA dimerization very likely through interaction with the kissing complex and converts it into a stable dimer. Furthermore, this NCp7-promoted conversion only occurs if the 240-280 stem-loop structure is present in HIV-1Lai RNA molecules and contains the autocomplementary G257CGCGC262 sequence. This study suggests that, under physiological conditions, an NCp7-mediated RNA conformational change is involved in the maturation of the HIV-1 RNA dimer.

Base Sequence↗

Anchoring hairpin ribozymes to long target RNAs by loop-loop RNA interactions.

Efficient ribozyme-mediated gene silencing requires the effective binding of a ribozyme to its specific target sequence. Stable stem-loop domains are key elements for efficiency of natural antisense RNAs. This work tests the possibility of using such naturally existing structural motifs for anchoring hairpin ribozymes when targeting long RNAs. Assays were performed with four catalytic antisense RNAs, based on the hairpin ribozyme (HP), that carried a stable stem-loop motif at their 3' end. Extensions consisted of one of the following motifs: the stem-loop II of the natural antisense RNA-CopA, its natural target in CopT, the TAR-RNA motif, or its complementary sequence alphaTAR. Interestingly, the presence of any of these antisense motifs resulted in an enhancement of catalytic performance against the ribozyme's 14-nucleotide-long target RNA (Swt). A series of artificial, long RNA substrates containing the Swt sequence and the natural TAR-RNA stem-loop were constructed and challenged with a catalytic antisense RNA carrying the TAR-complementary stem-loop. This cleaves each of these substrates significantly more efficiently than HP. The deletion of the TAR domain in the substrate, or its substitution by its complementary counterpart alphaTAR, abolishes the positive effect. These results suggest that the enhancement is owed to the interaction of both complementary stem-loop domains. Moreover, they demonstrate that the TAR domain can be used as an anchoring site to facilitate the access of hairpin ribozymes to their specific target sequences within TAR-containing RNAs.

Base Sequence↗

Stabilities of HIV-1 DIS type RNA loop-loop interactions in vitro and in vivo.

RNA loop-loop interactions are a prevalent motif in the formation of tertiary structure and are well suited to trigger molecular recognition between RNA molecules. We determined the stabilities of several loop-loop interactions with a constant 6 bp core sequence and varying unpaired flanking nucleotides and found that the flanking bases have a strong influence on the stability and ion dependence of the kissing complex. In general, the stabilities determined in 1 M Na+ are equivalent to those in the presence of near physiological Mg2+ concentrations. Therefore we further tested whether the stabilities determined in vitro and within yeast cells correlate, using a recently developed yeast RNA-hybrid system. For the majority of the loop types analyzed here, the melting temperatures determined in vitro are in good agreement with the relative beta-galactosidase activity in yeast cells, showing that data derived from in vitro measurements reflect in vivo properties. The most stable interactions are the naturally occurring HIV-1 DIS MAL and LAI derived loops with the motif (5' A(A)/(G)N6A 3'), emphasizing the crucial role of stable kissing complexes in HIV genome dimerization.

Base Pairing↗

DNA looping by two-site restriction endonucleases: heterogeneous probability distributions for loop size and unbinding force.

Proteins interacting at multiple sites on DNA via looping play an important role in many fundamental biochemical processes. Restriction endonucleases that must bind at two recognition sites for efficient activity are a useful model system for studying such interactions. Here we used single DNA manipulation to study sixteen known or suspected two-site endonucleases. In eleven cases (BpmI, BsgI, BspMI, Cfr10I, Eco57I, EcoRII, FokI, HpaII, NarI, Sau3AI and SgrAI) we found that substitution of Ca2+ for Mg2+ blocked cleavage and enabled us to observe stable DNA looping. Forced disruption of these loops allowed us to measure the frequency of looping and probability distributions for loop size and unbinding force for each enzyme. In four cases we observed bimodal unbinding force distributions, indicating conformational heterogeneity and/or complex binding energy landscapes. Measured unlooping events ranged in size from 7 to 7500 bp and the most probable size ranged from less than 75 bp to nearly 500 bp, depending on the enzyme. In most cases the size distributions were in much closer agreement with theoretical models that postulate sharp DNA kinking than with classical models of DNA elasticity. Our findings indicate that DNA looping is highly variable depending on the specific protein and does not depend solely on the mechanical properties of DNA.

Binding Sites↗

Specific in vivo labeling of cell surface-exposed protein loops: reactive cysteines in the predicted gating loop mark a ferrichrome binding site and a ligand-induced conformational change of the Escherichia coli FhuA protein.

The FhuA protein of Escherichia coli K-12 transports ferrichrome, the antibiotic albomycin, colicin M, and microcin 25 across the outer membrane and serves as a receptor for the phages T1, T5, phi80, and UC-1. FhuA is activated by the electrochemical potential of the cytoplasmic membrane, which probably opens a channel in FhuA. It is thought that the proteins TonB, ExbB, and ExbD function as a coupling device between the cytoplasmic membrane and the outer membrane. Excision of 34 residues from FhuA, tentatively designated the gating loop, converts FhuA into a permanently open channel. FhuA contains two disulfide bridges, one in the gating loop and one close to the C-terminal end. Reduction of the disulfide bridges results in a low in vivo reaction of the cysteines in the gating loop and no reaction of the C-terminal cysteines with biotin-maleimide, as determined by streptavidin-beta-galactosidase bound to biotin. In this study we show that a cysteine residue introduced into the gating loop by replacement of Asp-336 displayed a rather high reactivity and was used to monitor structural changes in FhuA upon binding of ferrichrome. Flow cytometric analysis revealed fluorescence quenching by ferrichrome and albomycin of fluorescein-maleimide bound to FhuA. Ferrichrome did not inhibit Cys-336 labeling. In contrast, labeling of Cys-347, obtained by replacing Val-347 in the gating loop, was inhibited by ferrichrome, but ferrichrome quenching was negligible. It is concluded that binding of ferrichrome causes a conformational change of the gating loop and that Cys-347 is part of or close to the ferrichrome binding site. Fluorescence quenching was independent of the TonB activity. The newly introduced cysteines and the replacement of the existing cysteines by serine did not alter sensitivity of cells to the FhuA ligands tested (T5, phi80, T1, colicin M, and albomycin) and fully supported growth on ferrichrome as the sole iron source. Since cells of E. coli K-12 display no reactivity to thiol reagents, newly introduced cysteines can be used to determine surface-exposed regions of outer membrane proteins and to monitor conformational changes during their function.

Amino Acid Sequence↗

Modified nucleotides of tRNA(3Lys) modulate primer/template loop-loop interaction in the initiation complex of HIV-1 reverse transcription.

In all retroviruses, reverse transcription is primed by a tRNA whose 3' end 18 nucleotides are complementary to the so called viral primer binding site. Previous work showed that reverse transcription of HIV-1 RNA is initiated by tRNA(3Lys). Using a variety of chemical and enzymatic structural probes, we investigated the interactions between HIV-1 RNA and its natural primer tRNA(3Lys). In addition to the predictable contacts between the viral primer binding site and the 3' end of tRNA(3Lys), a specific interaction takes place between an A-rich loop located upstream of the primer binding site region and the anticodon loop of tRNA(3Lys). This AAAA/Umcm5s2UUU loop-loop interaction is not observed when the natural primer is replaced by an in vitro synthesized tRNA(3Lys) transcript. Furthermore, dethiolation of the modified nucleotide mcm5s2U at position 34 of tRNA(3Lys) strongly destabilizes this interaction. Sequence and structure comparisons indicate that the primer/template loop-loop interaction is conserved in all HIV-1 isolates, and possibly also in HIV-2 and SIV.

Anticodon↗

Closure of transverse loop colostomy and loop ileostomy.

The aim of a defunctioning stoma is to protect patients from the consequences of faecal leakage and pelvic sepsis, should it occur. A retrospective audit of 77 patients who had undergone closure of a loop stoma between 1988 and 1996 was performed. Sixty patients had either transverse loop colostomy (52) or loop ileostomy (8) to defunction distal colorectal/anal anastomoses or pathology, and 17 patients had a loop ileostomy to defunction an ileoanal pouch. Those who had restorative proctocolectomy experienced a much higher (24%) complication rate than the loop colostomy group (5%), despite similar perioperative care and surgery performed by surgeons of equivalent seniority. The complication rate of ileostomy closure in pouch patients is similar to other published series. As a result of these findings, a selective approach to the use of loop ileostomy to protect pouches has been introduced. The absence of wound infections in our series would suggest that primary closure of the stomal wound without drainage can be achieved.

Adult↗

A LabVIEW model incorporating an open-loop arterial impedance and a closed-loop circulatory system.

While numerous computer models exist for the circulatory system, many are limited in scope, contain unwanted features or incorporate complex components specific to unique experimental situations. Our purpose was to develop a basic, yet multifaceted, computer model of the left heart and systemic circulation in LabVIEW having universal appeal without sacrificing crucial physiologic features. The program we developed employs Windkessel-type impedance models in several open-loop configurations and a closed-loop model coupling a lumped impedance and ventricular pressure source. The open-loop impedance models demonstrate afterload effects on arbitrary aortic pressure/flow inputs. The closed-loop model catalogs the major circulatory waveforms with changes in afterload, preload, and left heart properties. Our model provides an avenue for expanding the use of the ventricular equations through closed-loop coupling that includes a basic coronary circuit. Tested values used for the afterload components and the effects of afterload parameter changes on various waveforms are consistent with published data. We conclude that this model offers the ability to alter several circulatory factors and digitally catalog the most salient features of the pressure/flow waveforms employing a user-friendly platform. These features make the model a useful instructional tool for students as well as a simple experimental tool for cardiovascular research.

Animals↗

Closed-loop and open-loop accommodative vergence eye movements.

We examined accommodative vergence eye movements under conditions in which feedback about retinal-image slip was and was not present (i.e. closed-loop and open-loop conditions). We found that (a) in both conditions the two eyes started to move at the same time; (b) in the open-loop condition, vergence continued in both eyes; and (c) in the closed-loop condition, vergence continued in the occluded eye only, and the magnitude and velocity exceeded that of the occluded eye in the open-loop condition by a factor of two.

Accommodation, Ocular↗

Open-loop and closed-loop optokinetic nystagmus in squirrel monkeys (Saimiri sciureus) and in man.

Horizontal optokinetic nystagmus (OKN) was measured in 3 normal Squirrel monkeys by means of the electromagnetic search coil technique. Binocular and monocular stimulation of each eye to the left and right by moving vertical stripe patterns of 2.37 or 15 degree period were applied at angular velocities of 0.5 to 400 deg/s. After measurement of horizontal OKN under normal conditions, open-loop OKN gain was determined by monocular stimulation of an eyeball immobilized by means of retrobulbar injections of 11 units botulinum toxin (BoTx type A) and compared with the pre-injection data or with monocular stimulation of the other eye, which remained mobile. In normal Squirrel monkeys gain of optokinetic nystagmus reached values between 0.8 and 0.97 at angular velocities below 1.5 deg/s. Gain under these conditions was related to stimulus angular velocities Vs, i.e. Ge = Ve/Vs. A slightly higher gain was found for binocular than for monocular stimulation. No significant differences were found in OKN when monocular stimulation in the naso-temporal and in the temporo-nasal direction was applied. The upper cut-off angular velocity (-3 dB-point) reached values of 180-230 deg/s, significantly above those observed in man under similar stimulus conditions. Monocular optokinetic stimulation of an immobilized eye led to vigorous optokinetic nystagmus and OKAN of the other eye, whereby maximum gain (Gi = Ve/Vr) was found to be between 20 and 30 at lower retinal stimulus velocities (2-5 deg/s). Gain was related to retinal stimulus velocity Vr. Increase in Vr above 10 deg/s led to a decrease in gain with a slope of about 20 dB per decade. Measurement of gain of closed-loop OKN related to retinal stimulus velocity Vr (which was determined by the difference between Vs and Ve) led to a similar dependence of OKN as in closed-loop stimulus conditions. Differences in sensitivity between the temporal and the nasal visual hemifield stimulation evoking horizontal open-loop OKN are described. Directional selectivity appeared in these experiments. Open-loop OKN data from a human subject are reported. With highly attentive horizontal optokinetic gaze nystagmus, Ve depended on the duration of the pursuit phases of OKN. Ve accelerated with the duration of the individual slow phase of OKN and was reset by each backward saccade (of the covered mobile eye). OKN gain was considerably smaller when the subject intentionally pursued as many stripes as possible of the 1.15 degree period stripe pattern (gain related to Vr about 1.5-3).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Temporary colostomy in supralevator pelvic exenteration. A comparative study between stapled loop and loop colostomy.

A temporary loop colostomy is created in most patients undergoing total supralevator pelvic exenteration with low rectal anastomosis. We report 16 patients comparing two different types of loop colostomy. In half of them a normal loop colostomy was made and refashioning of the stoma was necessary in two patients because of faecal spillover in the presence of anastomotic leakage. In the other eight patients a functional terminal colostomy was created by application of a TA 55 stapler on the efferent loop. In this group no refashioning of the stoma was necessary and no difficulties were encountered on closure of this type of colostomy. Because of the high incidence of anastomotic leakage in this population, the stapled loop colostomy is the preferred method of choice because it eliminates any possible faecal spillover.

Adult↗

Dynamics of the flexible loop of triosephosphate isomerase: the loop motion is not ligand gated.

Using solid-state deuterium NMR, we have measured the motion of the flexible loop of triosephosphate isomerase (TIM) with and without substrate and transition-state analogs. The measurements were carried out on a catalytically competent mutant of TIM W90Y W157F containing a single tryptophan (W168) in the flexible loop; W168 is the only strictly conserved tryptophan in the currently available TIM sequences. The solid-state NMR samples were prepared by precipitation using polyethylene glycol, and kinetic analysis of the PEG-precipitated TIM gave values for kcat, Km, and KI similar to those measured in solution for the substrate and substrate and transition-state analogs. Deuterium NMR spectra of samples prepared with tryptophan labeled at the indole positions with and without any substrate or analogs indicate that the loop jumps between two conformations at a rate of 3 x 10(4) s-1 (from the predominant to the less populated form) with a population ratio of 10:1. Surprisingly, spectra of TIM ligated with a substrate analog, glycerol 3-phosphate (G3P), or with a tight-binding transition-state analog, phosphoglycolate (PGA), show that the loop moves with a rate similar to the rate in the empty enzyme and also has a similar population ratio for the two conformers. This observation indicates that loop closure is not ligand gated but is a natural motion of the protein. Furthermore, the measured rate is approximately matched to the turnover time. We did not observe a signal for TIM labeled with alpha-deuteriotryptophan, although it was prepared in a fashion analogous to the ring-labeled sample and had a specific activity and protein concentration comparable to the latter. For this deuterium concentration, we would expect to observe the NMR signal unless the deuterium relaxation were very slow. The hypothesis that the spin-lattice relaxation of the alpha-deuteron is very slow would be consistent with the observed dynamics of the ring-deuterated TIM.

Amino Acid Sequence↗

Mutational and proteolytic studies on a flexible loop in glutathione synthetase from Escherichia coli B: the loop and arginine 233 are critical for the catalytic reaction.

The function of the flexible loop which is disordered in crystal structure analysis of glutathione synthetase from Escherichia coli B has been investigated by limited proteolysis and kinetic measurements for the wild-type and mutant enzymes. Proteolysis of the intact enzyme using arginyl endopeptidase or trypsin brought about a time-dependent decrease in the enzymatic activity and the production of protein fragments. SDS-polyacrylamide gel electrophoresis and peptide sequence analysis showed that only a peptide bond between arginine 233 and glycine 234 in the loop was cleaved. Further, native polyacrylamide gel electrophoresis revealed that the cleaved enzyme retained almost the same quaternary structure as that of the wild-type enzyme. Upon protease treatment, the presence of substrates, ATP and/or gamma-L-glutamyl-L-cysteine (gamma-Glu-Cys), protected the loop from cleavage, but the presence of glycine was not capable of protecting it. In addition, replacement of arginine 233 in the loop with lysine by site-directed mutagenesis increased the Michaelis constants for gamma-Glu-Cys and glycine by factors of 28 and 213, respectively. The protection against cleavage on a similar protease incubation of this mutant enzyme was also observed in the presence of ATP and/or gamma-Glu-Cys, but the effect in the presence of both substrates was half as large as that for the wild-type enzyme. These results suggest that the loop covers the active site while ATP and gamma-Glu-Cys bind there and that it protects the unstable gamma-Glu-Cys phosphate intermediate from decomposition by bulk water.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Phosphorylation of the protein kinase C-theta activation loop and hydrophobic motif regulates its kinase activity, but only activation loop phosphorylation is critical to in vivo nuclear-factor-kappaB induction.

Protein kinase C (PKC)-theta, a member of the 'novel' subfamily of PKC isoforms, is of singular importance in transducing signals in T-lymphocytes. Since understanding of regulatory phosphorylation of novel PKCs is fragmentary and inconsistent with findings for 'classical' PKC isoforms, we investigated three potential phosphorylation sites on PKC-theta; in the activation loop (Thr(538)), turn motif (Ser(676)) and hydrophobic motif (Ser(695)). Combined evidence from phospho-specific antisera and MS demonstrates phosphorylation at all three sites. Unlike its closest paralogue, PKC-delta, lack of negative charge in the activation loop of PKC-theta results in a profound catalytic defect (>100-fold reduction in the T538A mutant); the high sequence similarity between PKC-theta and -delta assists in the formulation of structural hypotheses to account for this major difference. In contrast with mechanisms proposed for other PKC isoforms, phosphorylation at the other two sites does not reconstitute catalytic activity. Activation loop phosphorylation is critical in vivo, since the T538A mutant completely lost its capacity to mediate T-cell receptor-stimulation of nuclear factor kappaB (NF-kappaB) activation in Jurkat T-cells. Hydrophobic motif phosphorylation also substantially influences PKC-theta catalytic activity (5-fold reduction in the S695A mutant), but does not impair NF-kappaB activation in Jurkat T-cells. Its mechanism is independent of secondary effects on activation loop phosphorylation and cannot be explained by thermal instability. Turn motif phosphorylation has a limited effect on kinase activity, but negatively regulates other aspects of PKC-theta function, since the S676A mutant is more efficient than wild-type in inducing NF-kappaB activation in Jurkat T-cells. These findings expand our understanding of the roles of phosphorylation in novel PKCs, and indicate that PKC-theta is a constitutively competent kinase as a consequence of constitutive phosphorylation of its activation loop.

Amino Acid Motifs↗

A loop-loop "kissing" complex is the essential part of the dimer linkage of genomic HIV-1 RNA.

RNA-RNA interactions govern a number of biological processes. Several RNAs, including natural sense and antisense RNAs, interact by means of a two-step mechanism: recognition is mediated by a loop-loop complex, which is then stabilized by formation of an extended intermolecular duplex. It was proposed that the same mechanism holds for dimerization of the genomic RNA of human immunodeficiency virus type 1 (HIV-1), an event thought to control crucial steps of HIV-1 replication. However, whereas interaction between the partially self-complementary loop of the dimerization initiation site (DIS) of each monomer is well established, formation of the extended duplex remained speculative. Here we first show that in vitro dimerization of HIV-1 RNA is a specific process, not resulting from simple annealing of denatured molecules. Next we used mutants of the DIS to test the formation of the extended duplex. Four pairs of transcomplementary mutants were designed in such a way that all pairs can form the loop-loop "kissing" complex, but only two of them can potentially form the extended duplex. All pairs of mutants form heterodimers whose thermal stability, dissociation constant, and dynamics were analyzed. Taken together, our results indicate that, in contrast with the interactions between natural sense and antisense RNAs, no extended duplex is formed during dimerization of HIV-1 RNA. We also showed that 55-mer sense RNAs containing the DIS are able to interfere with the preformed HIV-1 RNA dimer.

Base Composition↗