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At least 55 records · Page 3Linked to original sources
PubMed2002

Function of the 90-loop (Thr90-Glu100) region of staphylokinase in plasminogen activation probed through site-directed mutagenesis and loop deletion.

Staphylokinsae (SAK) forms a bimolecular complex with human plasmin(ogen) and changes its substrate specificity by exposing new exosites that enhances accession of substrate plasminogen (PG) to the plasmin (Pm) active site. Protein modelling studies indicated the crucial role of a loop in SAK (SAK 90-loop; Thr(90)-Glu(100)) for the docking of the substrate PG to the SAK-Pm complex. Function of SAK 90-loop was studied by site-directed mutagenesis and loop deletion. Deletion of nine amino acid residues (Tyr(92)-Glu(100)) from the SAK 90-loop, resulted in approximately 60% reduction in the PG activation, but it retained the ability to generate an active site within the complex of loop mutant of SAK (SAKDelta90) and Pm. The preformed activator complex of SAKDelta90 with Pm, however, displayed a 50-60% reduction in substrate PG activation that remained unaffected in the presence of kringle domains (K1+K2+K3+K4) of PG, whereas PG activation by SAK-Pm complex displayed approximately 50% reduction in the presence of kringles, suggesting the involvement of the kringle domains in modulating the PG activation by native SAK but not by SAKDelta90. Lysine residues (Lys(94), Lys(96), Lys(97) and Lys(98)) of the SAK 90-loop were individually mutated into alanine and, among these four SAK loop mutants, SAK(K97A) and SAK(K98A) exhibited specific activities about one-third and one-quarter respectively of the native SAK. The kinetic parameters of PG activation of their 1:1 complex with Pm indicated that the K(m) values of PG towards the activator complex of these two SAK mutants were 4-6-fold higher, suggesting the decreased accessibility of the substrate PG to the activator complex formed by these SAK mutants. These results demonstrated the involvement of the Lys(97) and Lys(98) residues of the SAK 90-loop in assisting the interaction with substrate PG. These interactions of SAK-Pm activator complex via the SAK 90-loop may provide additional anchorage site(s) to the substrate PG that, in turn, may promote the overall process of SAK-mediated PG activation.

Base Sequence↗
PubMed1982

D-loop cycle. A circular reaction sequence which comprises formation and dissociation of D-loops and inactivation and reactivation of superhelical closed circular DNA promoted by recA protein of Escherichia coli.

Excess recA protein, a protein essential to general genetic recombination in Escherichia coli, promotes a sequence of formation and dissociation of D-loops from negative superhelical closed circular double-stranded DNA (form I DNA) and homologous single-stranded fragments in the presence of excess ATP, resulting in inactivation of the form I DNA without apparent damage to the DNA. The dissociation of D-loops is accompanied by hydrolysis of ATP to ADP that apparently depends on homologous DNA molecules (homology-dependent ATP hydrolysis). However, at a lower concentrations of ATP, we observed anomalous kinetics in the formation and dissociation of D-loops; as the concentration of ATP was decreased, there was a progressively smaller dissociation of D-loops and a faster resynthesis in the second phase, without changing the rate of the first formation of D-loops. This anomaly might suggest that, as the increase in the amount of ADP relative to that of ATP, dissociation form I DNA is stimulated before formation of D-loops is inhibited. We found that addition of ADP inhibited competitively both formation and dissociation of D-loops and that the latter process was more sensitive to the inhibition than was the former process. Addition of a sufficient amount of ADP to inhibit both formation and dissociation of D-loops, cessation of homology-dependent hydrolysis of ATP, or incubation at low temperature resulted in reactivation of form I DNA that had been inactivated by the sequence. In the presence of an ATP-regenerating system, we confirmed our previous result that limiting the amount of recA protein also causes anomalous kinetics in the formation and dissociation of D-loops. These observations indicate that the formation and dissociation of D-loops and the inactivation and reactivation of form I DNA make a circular reaction sequence.

Bacterial Proteins↗
PubMed1995

The effects of spaceflight on open-loop and closed-loop postural control mechanisms: human neurovestibular studies on SLS-2.

Stabilogram-diffusion analysis was used to examine how prolonged periods in microgravity affect the open-loop and closed-loop postural control mechanisms. It was hypothesized that following spaceflight: (1) the effective stochastic activity of the open-loop postural control schemes in astronauts is increased; (2) the effective stochastic activity and uncorrelated behavior, respectively, of the closed-loop postural control mechanisms in astronauts are increased; and (3) astronauts utilized open-loop postural controls schemes for shorter time intervals and smaller displacements. Four crew members and two alternates from the 14-day Spacelab Life Sciences 2 Mission were included in the study. Each subject was tested under eyes-open, quiet-standing conditions on multiple preflight and postflight days. The subjects' center-of-pressure trajectories were measured with a force platform and analyzed according to stabilogram-diffusion analysis. It was found that the effective stochastic activity of the open-loop postural control schemes in three of the four crew members was increased following spaceflight. This result is interpreted as an indication that there may be in-flight adaptations to higher-level descending postural control pathways, e.g., a postflight increase in the tonic activation of postural muscles. This change may also be the consequence of a compensatory (e.g., "stiffening") postural control strategy that is adopted by astronauts to account for general feeling of postflight unsteadiness. The crew members, as a group, did not exhibit any consistent preflight/postflight differences in the steady-state behavior of their closed-loop postural control mechanisms or in the functional interaction of their open-loop and closed-loop postural control mechanisms. These results are interpreted as indications that although there may be in-flight adaptations to the vestibular system and/or proprioceptive system, input from the visual system can compensate for such changes during undisturbed stance.

Humans↗
PubMed2006

Usefulness of T-wave loop and QRS complex loop to predict mortality after acute myocardial infarction.

The aim of the study was to assess whether parameters based on the T-wave loop and QRS loop predict mortality, and cardiac mortality in particular, during follow-up of consecutive survivors of acute myocardial infarction (AMI). Patients with AMI (n = 437), treated according to contemporary guidelines, underwent digital high-resolution electrocardiography in orthogonal Frank leads (X, Y, Z) 5 to 14 days after AMI. Several T-wave and QRS loop parameters, such as the width and height of the loops and their ratio, T-wave loop dispersion (TWLD), QRS loop dispersion, and co-sine of the angle between the main vectors of the T-wave and QRS loops (TCRT), were calculated using a custom-made software package. During an average follow-up period of 43 +/- 14 months, 53 patients (12%) died. Of these 53 deaths, 35 were cardiac. TWLD and TCRT were the T-wave loop/QRS loop parameters that best predicted for cardiac mortality on univariate comparison (35.4 +/- 5.62 vs 32.8 +/- 2.87 for TWLD, p < 0.001 and -0.135 +/- 0.665 vs -0.657 +/- 0.518 for TCRT, p < 0.001, alive vs cardiac death, respectively). After adjustment for clinical risk markers in the Cox regression analysis, TWLD still significantly predicted for cardiac mortality (p < 0.05); however, TCRT had lost its predictive power. TWLD did not have significant univariate or multivariate association with noncardiac mortality. In conclusion, TWLD that describes the shape of the T-wave loop is a specific predictor of cardiac death and independent of the clinical risk markers in the current treatment era of patients with AMI.

Aged↗
PubMed1997

Optimizing the metal binding parameters of an EF-hand-like calcium chelation loop: coordinating side chains play a more important tuning role than chelation loop flexibility.

In calcium signaling pathways regulated by the EF-hand Ca2+ binding motif, proper regulation requires that the equilibrium and kinetics of Ca2+ binding to the EF-hand chelation loop be precisely optimized for each physiological application. Studies of small-molecule organic chelators have shown that metal binding parameters can be tuned both by the nature of the coordinating ligands and by the structural framework to which these ligands are attached. By analogy, the present study tests the relative importance of (i) coordinating side chains and (ii) backbone torsion angle constraints to the tuning of an EF-hand-like Ca2+ chelation loop. A series of engineered chelation loops are generated by modifying Ca2+ binding site of the Escherichia coli galactose binding protein. The resulting loops, each containing an altered coordinating side chain or a Gly substitution, are compared with respect to their metal binding affinities, specificities, and dissociation kinetics. The Gly variants examined include substitutions which eliminate or introduce a Gly at each of the nine chelation loop positions. The results reveal that Gly is not tolerated at loop positions 1, 3, 5, or 8 or at the external coordinating position, where the removal of a key coordinating or hydrophobic side chain destabilizes the protein. In contrast, Gly residues at loop positions 2, 4, 6, and 7, none of which is required for side chain coordination, have little effect on Ca2+ affinity and the ability to discriminate between cations of different size and charge. Kinetic measurements show that some of these Gly residues measurably alter the rates of metal ion association and dissociation, but in each case the two rates are changed by approximately the same factor so that the effects on equilibrium are minor. Overall, Gly residues yield surprisingly small effects at loop positions 2, 4, 6, and 7, especially when compared to the larger equilibrium and kinetic effects observed for coordinating side chain substitutions. It follows that the conserved Gly at position 6 is not required for Ca2+ binding and that constraints on the backbone torsion angles at the non-coordinating side chain positions 2, 4, 6, and 7 play a relatively minor role in tuning metal binding parameters. Instead, specific coordinating side chains optimize the metal binding parameters of the GBP chelation loop for its protein context and biological application.

Calcium↗
PubMed2002

Randomized clinical trial comparing loop ileostomy and loop transverse colostomy for faecal diversion following total mesorectal excision.

BACKGROUND: The aim of this study was to compare loop ileostomy and loop transverse colostomy as the preferred mode of faecal diversion following low anterior resection with total mesorectal excision for rectal cancer. METHODS: Patients who required proximal diversion after low anterior resection with total mesorectal excision were randomized to have either a loop ileostomy or a loop transverse colostomy. Postoperative morbidity, stoma-related problems and morbidity following closure were compared. RESULTS: From April 1999 to November 2000, 42 patients had a loop ileostomy and 38 had a loop transverse colostomy constructed following low anterior resection. Postoperative intestinal obstruction and prolonged ileus occurred more commonly in patients with an ileostomy (P = 0.037). There was no difference in time to resumption of diet, length of hospital stay following stoma closure and incidence of stoma-related complications after discharge from hospital. A total of seven patients had intestinal obstruction from the time of stoma creation to stoma closure (six following ileostomy and one following colostomy; P = 0.01). CONCLUSION: Intestinal obstruction and ileus are more common after loop ileostomy than loop colostomy. Loop transverse colostomy should be recommended as the preferred method of proximal faecal diversion.

Adult↗
PubMed1996

Loop replacement and random mutagenesis of omega-loop D, residues 70-84, in iso-1-cytochrome c.

To study the role of omega loop D, residues 70-84, in the structure and function of yeast iso-1-cytochrome c, this loop was replaced with homologous and heterologous loops. A novel method was developed for rapid insertion of these mutations into the yeast chromosome at the CYC1 locus. The strains containing these loop replacement cytochromes cannot grow on nonfermentable carbon sources, indicating that the proteins are nonfunctional. Whole cell difference spectroscopy shows that no holocytochrome c is present; however, apoprotein is found by immunoblot analysis. Thus, apoprotein is present in these mutant strains, but it cannot bind heme and cannot compete with wild type apoprotein conversion to holoprotein. This is a unique example of a set of loop replacements that do not produce folded protein, and these results suggest that the loop D amino acid sequence in iso-1-cytochrome c plays a significant role in cytochrome c biosynthesis in vivo. To identify the significant amino acids in loop D, random mutagenesis of six highly conserved loop residues, Tyr-74, Ile-75, Pro-76, Gly-77, Thr-78, and Lys-79, was accomplished. Sequencing of the random mutants shows that strict conservation of none of these residues is required to produce a minimally functional cytochrome c. Preferences are found for small, hydrophilic or aromatic residues at position 74, hydrophobic residues at position 75, glycine and arginine at positions 76 and 77, and beta-branched amino acids at position 78. Implications for the role of loop D in the structure and function of iso-1-cytochrome c are discussed.

Amino Acid Sequence↗
PubMed1997

Molecular tuning of an EF-hand-like calcium binding loop. Contributions of the coordinating side chain at loop position 3.

Calcium binding and signaling orchestrate a wide variety of essential cellular functions, many of which employ the EF-hand Ca2+ binding motif. The ion binding parameters of this motif are controlled, in part, by the structure of its Ca2+ binding loop, termed the EF-loop. The EF-loops of different proteins are carefully specialized, or fine-tuned, to yield optimized Ca2+ binding parameters for their unique cellular roles. The present study uses a structurally homologous Ca2+ binding loop, that of the Escherichia coli galactose binding protein, as a model for the EF-loop in studies examining the contribution of the third loop position to intramolecular tuning. 10 different side chains are compared at the third position of the model EF-loop with respect to their effects on protein stability, sugar binding, and metal binding equilibria and kinetics. Substitution of an acidic Asp side chain for the native Asn is found to generate a 6,000-fold increase in the ion selectivity for trivalent over divalent cations, providing strong support for the electrostatic repulsion model of divalent cation charge selectivity. Replacement of Asn by neutral side chains differing in size and shape each alter the ionic size selectivity in a similar manner, supporting a model in which large-ion size selectivity is controlled by complex interactions between multiple side chains rather than by the dimensions of a single coordinating side chain. Finally, the pattern of perturbations generated by side chain substitutions helps to explain the prevalence of Asn and Asp at the third position of natural EF-loops and provides further evidence supporting the unique kinetic tuning role of the gateway side chain at the ninth EF-loop position.

Calcium-Binding Proteins↗
PubMed2003

Loops In Proteins (LIP)--a comprehensive loop database for homology modelling.

One of the most important and challenging tasks in protein modelling is the prediction of loops, as can be seen in the large variety of existing approaches. Loops In Proteins (LIP) is a database that includes all protein segments of a length up to 15 residues contained in the Protein Data Bank (PDB). In this study, the applicability of LIP to loop prediction in the framework of homology modelling is investigated. Searching the database for loop candidates takes less than 1 s on a desktop PC, and ranking them takes a few minutes. This is an order of magnitude faster than most existing procedures. The measure of accuracy is the root mean square deviation (RMSD) with respect to the main-chain atoms after local superposition of target loop and predicted loop. Loops of up to nine residues length were modelled with a local RMSD <1 A and those of length up to 14 residues with an accuracy better than 2 A. The results were compared in detail with a thoroughly evaluated and tested ab initio method published recently and additionally with two further methods for a small loop test set. The LIP method produced very good predictions. In particular for longer loops it outperformed other methods.

Data Interpretation, Statistical↗
PubMed2006

Mutation of Gly51 to serine in the P-loop of Lactobacillus casei folylpolyglutamate synthetase abolishes activity by altering the conformation of two adjacent loops.

Based upon the three-dimensional structure of Lactobacillus casei folylpolyglutamate synthetase (FPGS), site-directed mutagenesis studies were performed on three residues associated with the ATPase site: Gly51, Ser52 and Ser73. Gly51 and Ser52 are at the end of the P-loop, which is involved in triphosphate binding. A G51S mutant enzyme and a G51S/S52T double-mutant enzyme were made in order to alter the FPGS P-loop to more closely resemble the sequences found in other ATPase and GTPase enzymes. Ser73 is on a neighboring loop (the Omega-loop) and precedes a proline residue found to be in a cis conformation. The carbonyl O atom of Ser73 is one of the protein ligands for the essential Mg(2+) ion involved in ATP binding and hydrolysis and the Omega-loop is involved in binding the folate substrate 5,10-methylenetetrahydrofolate. The serine residue was mutated to alanine and this is the only one of the three mutants which retains some FPGS activity. The structures of the G51S, G51S/S52T and S73A mutant proteins have been solved to high resolution, along with the structure of the apo wild-type FPGS. The P-loop in both the G51S and G51S/S52T mutant proteins remains unaltered, yet both structures show a large conformational rearrangement of the Omega-loop in which a cis-Pro residue has switched conformation to a trans-peptide. The structure of the Omega-loop is severely disrupted and as a consequence structural rearrangements are observed in the peptide linker joining the two domains of the enzyme. Magnesium binding in the active site is also disrupted by the presence of the serine side chain at position 51 and by the repositioning of the carbonyl O atom of Ser73 and a water molecule is bound in place of the Mg(2+) ion. The S73A mutant protein retains the cis-Pro configuration in the Omega-loop and the Mg(2+) site remains intact. The cis-Pro is also observed in the structure of the substrate-free form of FPGS (apoFPGS), maintained in the absence of Mg(2+) by a hydrogen-bonding network involving water molecules in the active site. It is only in the complete absence of water or Mg(2+) in the binding site that the cis-Pro switches to the trans conformation.

Amino Acid Sequence↗
PubMedDate not supplied

The three-loop pulley suture versus two locking-loop sutures for the repair of canine achilles tendons.

OBJECTIVE: To compare a 3-loop pulley suture pattern with 2 locking-loop sutures for the repair of components of the canine Achilles mechanism. STUDY DESIGN: In vitro biomechanical study. ANIMALS: Forty-eight paired tendons collected from 9 canine cadavers. METHODS: Paired tendons were repaired with either a 3-loop pulley suture or 2 locking-loop sutures and tensile tested to failure. To ensure accurate anastomosis gap measurement a direct, non-contact, method of gap measurement, using digital video, was devised. Load initiating gap formation (defined as load at a 1 mm gap) and load producing a 3 mm gap were evaluated in addition to maximum load, gap at failure, mode of failure, and time spent placing the sutures. RESULTS: Maximum load values were similar for both repairs. The mean 1 mm gap loads were 44.0 and 18.4 N, and the mean 3 mm gap loads were 56.3 and 34.7 N, for the 3-loop pulley pattern and the 2 locking-loops, respectively; these differences were statistically significant. The 3-loop pulley pattern was faster to place and resulted in a smaller gap at failure. All but 2 repairs failed by suture pull out. CONCLUSIONS: The 3-loop pulley pattern is more resistant to gap formation during tensile loading, and is quicker to place, than 2 locking-loop sutures. CLINICAL RELEVANCE: Gap formation can significantly delay tendon healing. Tendon repairs with a gap >3 mm are reported to be at increased risk of rupture during the first 6 weeks postoperatively.

Achilles Tendon↗
PubMed2001

Role of stem B, loop B, and nucleotides next to the primer binding site and the kissing-loop domain in human immunodeficiency virus type 1 replication and genomic-RNA dimerization.

Stem-loop B is a 12-nucleotide [nt]-long completely conserved sequence postulated to form a 4-bp stem and a 4-nt internal loop under the kissing-loop hairpin (klh) (nt 248 to 270) of human immunodeficiency virus type 1 (HIV-1) genomic RNA. We investigated its role in viral replication, genomic RNA dimerization, and dimerization of partial HIV-1 RNA transcripts. The putative CUCG246-CGAG277 duplex was replaced by nine alternative complementary sequences, five likely to base pair only in short RNAs and four likely to base pair in long (approximately 500-nt) RNAs, as assessed by the algorithm mfold. Among the five former sequences, none preserved genome dimerization and all reduced viral replication by 98 to 99.9%. Among the four latter sequences, three (MB6, -9, and -10) preserved genome dimerization, one (MB7) did not significantly inhibit it, and two (MB9 and -10) preserved viral replication. We conclude that duplex formation by stem B nucleotides is necessary for viral infectivity and complete genome dimerization. Deleting the 5' or 3' side of loop B or of stem B had little impact on dimerization of partial RNA transcript and no impact on klh folding (and, for loop B mutations, on stem B folding), but each deletion inhibited genome dimerization almost as much as klh destruction. This suggests that loop B is required for complete genome dimerization and that loop B and stem B stimulate dimerization only in very long RNAs and/or in the presence of unidentified viral and cellular factors. Finally, we asked if nine deletions or nucleotide substitutions within nt 200 to 242 and/or nt 282 to 335 could influence genome dimerization. These mutations had intermediate inhibitory impacts consistent with their predicted influence on stem B, loop B, and klh formation. Two exceptions were Delta200-226 and Delta236-242 genomic RNAs, which dimerized relatively poorly despite having neutral or positive influences on stem B, loop B, and klh folding.

Animals↗
PubMed1990

Furosemide acts on short loop of descending thin limb, but not on long loop.

In order to elucidate the tubular sites of action of loop diuretics such as furosemide, bumetanide and ethacrynic-cysteine complex within isolated rat descending thin limbs, cellular ATP was measured by luciferin-luciferase technique. When short descending thin limbs of Henle's loop (SDL) were incubated in the absence of exogenous substrate at 37 degrees C, cellular ATP content was decreased in a time-dependent manner (up to 49% after 60 min). This ATP decrease, however, was retarded significantly in the presence of loop diuretics at 60 min. The mean percentage of change in ATP compared with the control for each loop diuretic in SDL was as follows: 10(-5) M furosemide, 178%; 10(-5) M bumetanide, 189%; and 10(-7) M ethacrynic-cysteine complex, 154%; respectively. To the contrary, cellular ATP in long descending thin limb of Henle's loop (LDL) was not changed by loop diuretics compared with the control. A similar protection against ATP depletion was observed in the medullary thick ascending limb of Henle's loop, in which the mean percentage was as follows: 10(-5) M furosemide, 163%; 10(-5) M bumetanide, 187%; and 10(-7) M ethacrynic-cysteine complex, 134%. Similarly to LDL, the cellular ATP did not change in outer medullary collecting tubule. From these results, we conclude that loop diuretics act on the isolated rat SDL, but not on LDL.

Adenosine Triphosphate↗
PubMed2026

R-loops and D-loops: a delicate balance in genomic stability and instability.

R-loops and D-loops are three-stranded nucleic acid structures that have emerged as central regulators of genome stability, gene expression, and DNA metabolism. R-loops form co-transcriptionally or post-transcriptionally when nascent RNA re-anneals with the template DNA strand, generating an RNA: DNA hybrid that displaces the non-template strand into a single-stranded state. These structures are enriched at CpG island promoters, transcription termination sites, and immunoglobulin class-switch regions, where they coordinate transcription regulation, chromatin remodeling, and DNA damage signaling. D-loops are formed when a single-stranded DNA segment pairs with one strand of a duplex and displaces the other, arising through context-dependent mechanisms that include RAD51- or DMC1-mediated strand invasion in homologous recombination, shelterin-assisted invasion at telomeres, and replication-coupled strand displacement at the mitochondrial DNA origin. They serve as indispensable intermediates in double-strand break repair, telomere maintenance, and mitochondrial DNA replication. Recent cryo-electron microscopy studies have resolved the stepwise RAD51-mediated strand exchange mechanism at near-atomic resolution, substantially advancing structural understanding of D-loop biogenesis. Despite their differences in molecular composition, both structures remodel Watson-Crick base pairing and, when dysregulated, are associated with replication fork stalling, transcription-replication conflicts, and aberrant recombination. This review systematically compares the structural features, formation mechanisms, regulatory networks, and biological functions of R-loops and D-loops, with emphasis on their convergent roles in safeguarding genome integrity. We further discuss rapidly evolving detection technologies and emerging therapeutic strategies targeting these structures in cancer and neurodegeneration, identifying key unresolved questions for future investigation.

Genomic Instability↗
PubMed1987

Structural features of the terminal loop region of frog retinal rod outer segment disk membranes: II. Organization of the terminal loop complex.

In addition to a lipid bilayer component (Corless, Fetter, and Costello: J. Comp. Neurol. 257:1-8, '87), the terminal loop region of frog rod outer segment (ROS) disks displays a clustering of discrete elements referred to as the terminal loop complex. It consists of (1) semicircular or crescentic densities within the terminal loop, (2) linear interdisk densities spanning the cytoplasm near terminal loops, and (3) distinctive freeze-fracture particles associated with the terminal loop, located between 1 and 2. The linear interdisk densities are organized on a two-dimensional lattice that appears to ensheath completely the lamellar domains of all ROS disks. Indirect evidence is presented for a net axial alignment of intraloop densities. We interpret the large freeze-fracture particles of the terminal loop region to reflect transmembrane components that connect the interdisk and intraloop densities. Thus, we propose that the entire terminal loop (TL) complex is organized on a two-dimensional net. We further infer that each TL complex is organized as a dimeric unit and that such dimers interact axially and laterally to generate the observed lattice structure. It is suggested that one component of the terminal loop complex is the high molecular weight protein localized along the disk perimeter by Papermaster, Schneider, Zorn, and Kraehenbuhl (J. Cell. Biol. 78:415-425, '78).

Animals↗
PubMed2000

Open-loop and closed-loop optokinetic nystagmus (OKN) in myasthenia gravis and nonmyasthenic subjects.

Optokinetic nystagmus (OKN) eye movements of myasthenia gravis (MG) and nonmyasthenic ocular palsies, and normal subjects were examined under closed-loop and open-loop conditions. The open-loop OKN condition was achieved by adding the signal of eye-movement velocity of OKN to the computer-generated signal controlling the stimulus grating moving. The OKN was recorded by means of electromagnetic search scleral coil technique. In MG patients, the open-loop gains of OKN increased significantly after the intramuscular injection of an acetylcholinesterase inhibitor, neostigmine, while the closed-loop OKN gains were not significantly changed. Both the closed-loop and open-loop OKN gains of normal subjects and nonmyasthenic patients were not increased for the administration of neostigmine. The experimental results indicated that the open-loop OKN gain could be sensitive to reflect the changes of the function of neuromuscular junction in MG patients.

Adolescent↗
PubMed2001

Solution-state NMR investigations of triosephosphate isomerase active site loop motion: ligand release in relation to active site loop dynamics.

Product release is partially rate determining in the isomerization reaction catalyzed by Triosephosphate Isomerase, the conversion of dihydroxyacetone phosphate to D-glyceraldehyde 3-phosphate, probably because an active-site loop movement is necessary to free the product from confinement in the active-site. The timescale of the catalytic loop motion and of ligand release were studied using 19F and 31P solution-state NMR. A 5'-fluorotryptophan was incorporated in the loop N-terminal hinge as a reporter of loop motion timescale. Crystallographic studies confirmed that the structure of the fluorinated enzyme is indistinguishable from the wild-type; the fluorine accepts a hydrogen bond from water and not from a protein residue, with minimal perturbation to the flexible loop stability. Two distinct loop conformations were observed by 19F NMR. Both for unligated (empty) and ligated enzyme samples a single species was detected, but the chemical shifts of these two distinct species differed by 1.2 ppm. For samples in the presence of subsaturating amounts of a substrate analogue, glycerol 3-phosphate, both NMR peaks were present, with broadened lineshapes at 0 degrees C. In contrast, a single NMR peak representing a rapid average of the two species was observed at 30 degrees C. We conclude that the rate of loop motion is less than 1400 s(-1) at 0 degrees C and more than 1400 s(-1) at 30 degrees C. Ligand release was studied under similar sample conditions, using 31P NMR of the phosphate group of the substrate analogue. The rate of ligand release is less than 1000 s(-1) at 0 degrees C and more than 1000 s(-1) at 30 degrees C. Therefore, loop motion and product release are probably concerted and likely to represent a rate limiting step for chemistry.

Binding Sites↗
PubMed2002

Accelerated reaction by loop-mediated isothermal amplification using loop primers.

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity. We have developed a method that accelerates the LAMP reaction by using additional primers, termed loop primers. Loop primers hybridize to the stem-loops, except for the loops that are hybridized by the inner primers, and prime strand displacement DNA synthesis. Although both inner and loop primers react via the loops, they do so by different mechanisms. The LAMP method presented here uses loop primers to achieve reaction times of less than half that of the original LAMP method. Since the total time of analysis including detection is less than 1h, this new method should facilitate genetic analysis, including genetic diagnosis in the clinical laboratory.

Base Sequence↗