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PubMed1997

Predicting the conformational class of short and medium size loops connecting regular secondary structures: application to comparative modelling.

Loops are regions of non-repetitive conformation connecting regular secondary structures. They are both the most difficult and error prone regions of a protein to solve by X-ray crystallography and the hardest regions to model using comparative procedures. Although a loop can sometimes be modelled from a homologue, very often it must be selected from outside the family. The loop prediction procedure, SLoop, attempts to identify the conformational class of the loop rather than to select a specific loop from a set of fragments extracted from known structures or generated ab initio. Templates are constructed for each of the 161 loop conformational classes that have been identified from the clustering of the structures of some 2024 loops of one to eight residues in length. A class template describes both sequence preferences and relative disposition of bounding secondary structures. During comparative modelling, the conformation of a loop can be predicted by identifying a loop class with which its sequence and disposition of bounding secondary structures are compatible. The procedure is tested on an unrelated non-redundant set of 1785 loops under stringent and lax evaluation schemes. Optimal sequence score cut-offs are identified such that the prediction rate is equal to the percentage of loops assigned to acceptable classes. Under the stringent evaluation, at the optimal sequence score cut-off, a conformation is predicted for 50% of loops of which 47% are correct, while under the lax evaluation a conformation is predicted for 63% of loops of which 54% are correct. Sequence score is shown to be a good indicator of the probability of a prediction being correct. Loop length also has a strong affect on prediction outcomes. Considering only loops of two to five residues in length, under the stringent evaluation 62% of loops are predicted with 52% of these predictions being correct while under the lax evaluation predictions are provided for 75% of loops of which 57% are correct.

Computer Simulation↗

PubMed1997

The structure of an RNA "kissing" hairpin complex of the HIV TAR hairpin loop and its complement.

We have used nuclear magnetic resonance (NMR) to obtain the structure of an RNA "kissing" hairpin complex formed between the HIV-2 TAR hairpin loop and a hairpin with a complementary loop sequence. Kissing hairpins are important in natural antisense reactions; their complex is a specific target for protein binding. The complex has all six nucleotides of each loop paired to form a bent quasicontinuous helix of three coaxially stacked helices: two stems plus a loop-loop interaction helix. Experimental constraints derived from heteronuclear and homonuclear NMR data on 13C and 15N-labeled RNA led to a structure for the loop-loop helix with an average root-mean-square deviation of 0.83 (+/-0.10) A for 33 converged structures relative to the average structure. The loop-loop helix of the kissing complex is distorted compared to A-form RNA. Its major groove is blocked by the phosphodiester bonds that connect the first loop residue of each hairpin with its own stem, and it is flanked by two negatively charged phosphate clusters. The loop-loop helix has alternating helical twists between adjacent base-pairs. The base-pairs at the helix junctions are overwound and three base-pairs near the helix junctions adopt high propeller twists. All these changes reduce the distance needed for the bridging phosphodiester bonds connecting each stem and loop to cross the major groove of the loop-loop helix, and result in a deformed RNA helix with localized perturbations in the minor groove surface. The alternating helical twist pattern, plus other distortions in the loop-loop helix may be important for Rom protein recognition of the kissing hairpin complex.

Bacterial Proteins↗

PubMed1990

Perturbation of DNA hairpins containing the EcoRI recognition site by hairpin loops of varying size and composition: physical (NMR and UV) and enzymatic (EcoRI) studies.

We have investigated loop-induced structural perturbation of the stem structure in hairpins d(GAATTCXnGAATTC) (X = A, T and n = 3, 4, 5 and 6) that contain an EcoRI restriction site in close proximity to the hairpin loop. Oligonucleotides containing either a T3 or a A3 loop were not hydrolyzed by the restriction enzyme and also showed only weak binding to EcoRI in the absence of the cofactor Mg2+. In contrast, hairpins with larger loops are hydrolyzed by the enzyme at the scission site next to the loop although the substrate with a A4 loop is significantly more resistant than the oligonucleotide containing a T4 loop. The hairpin structures with 3 loop residues were found to be thermally most stable while larger hairpin loops resulted in structures with lower melting temperatures. The T-loop hairpins are thermally more stable than the hairpins containing the same number of A residues in the loop. As judged from proton NMR spectroscopy and the thermodynamic data, the base pair closest to the hairpin loop did form in all cases studied. The hairpin loops did, however, affect the conformation of the stem structure of the hairpins. From 31P and 1H NMR spectroscopy we conclude that the perturbation of the stem structure is stronger for smaller hairpin loops and that the extent of the perturbation is limited to 2-3 base pairs for hairpins with T3 or A4 loops. Our results demonstrate that hairpin loops modulate the conformation of the stem residues close to the loop and that this in turn reduces the substrate activity for DNA sequence specific proteins.

DNA↗

PubMed2007

Importance of A-loop complementarity with tRNAHis anticodon for continued selection of tRNAHis as the HIV reverse transcription primer.

BACKGROUND: Human immunodeficiency virus (HIV-1) preferentially selects tRNALys,3 as the primer for reverse transcription. HIV-1 can be forced to select alternative tRNAs through mutation in the primer-binding site (PBS) and a region upstream of the PBS designated as the A-loop. Alteration of the PBS and A-loop to be complementary to the 3' terminal nucleotides and anticodon of tRNAHis results in HIV-1 that can stably utilize this tRNA for replication. RESULTS: In the current study, we have investigated the effect that mutations within the A-loop have on the stability of HIV-1 with a PBS complementary to tRNAHis. For these studies, we have altered the A-loop to be complementary to tRNAMet, tRNAGln, tRNAIle, tRNAThr and tRNASer. All substitutions of the A-loops with the PBS complementary to tRNAHis resulted in a reduction of infectious virus obtained following transfection of proviral genomes in the 293T cells. Virus replication in SupT1 cells was also impaired as a result of the alteration of the A-loop. Viruses with the A-loop complementary to tRNALys,3 and tRNASer reverted to utilize tRNALys,3 following in vitro replication. In contrast, viruses with the A-loop complementary to the other tRNAs remained stable and continued to use tRNAHis. RNA modeling of the stem-loop structure revealed that nucleotides were displayed on the loop region that could potentially interact with the anticodon of tRNAHis. To further explore the effects of the A-loop mutations on virus replication, the A-loops complementary to tRNASer or tRNAHis were cloned into the wild type genome with the PBS complementary to tRNALys,3. Transfection of proviral genomes which contained the wild type PBS and A-loops complementary to tRNASer or tRNAHis into 293 T cells did not impact on the production of viruses as measured by p24 antigen ELISA. However, viruses with the A-loop complementary to tRNAHis had greatly reduced infectivity and replicated poorly in SupT1 compared to the wild type or viruses with the A-loop complementary to tRNASer. CONCLUSION: These studies demonstrate that complementarity of A-loop region with the anticodon of tRNAHis has a pronounced effect on the capacity of HIV-1 to utilize tRNAHis as the primer for reverse transcription. Complementarity between A-loop and anticodon of the tRNA then is important for the selection of the tRNA primer used for reverse transcription.

Anticodon↗

PubMed1992

Studies of DNA dumbbells. II. Construction and characterization of DNA dumbbells with a 16 base-pair duplex stem and Tn end loops (n = 2, 3, 4, 6, 8, 10, 14).

The preparation and characterization of DNA dumbbells that contain the 16 base-pair duplex sequences 5'G-C-A-T-A-G-A-T-G-A-G-A-A-T-G-C3' (set 1) and 5'G-C-A-T-C-A-T-C-G-A-T-G-A-T-G-C3' (set 2) are reported. The dumbbells of set 1 have the duplex stem nucleated on both ends by Tn (n = 2, 3, 4, 6, 8, 10, and 14) loops. The dumbbells of set 2 have Tn (n = 2, 4, 8, 10) end loops. For the molecules of set 1, effects of end loop size on the electrophoretic mobility, CD and UV absorbance spectra, and cleavage by restriction enzymes, were investigated. Effects of loop size on the CD spectra and restriction enzyme cleavage of the molecules of set 2 were also examined. Optical melting curves of the molecules of set 1 were collected as a function of sodium ion concentration from 30 to 120 mM. These investigations revealed that as loop size decreases, the electrophoretic mobilities, rates of enzyme cleavage, and optical melting temperatures increase. For end loops with at least three T's the observed increases are inversely proportional to loop size. The behavior of the dumbbell with T2 end loops departs from this linear dependence and is anomalous in every experimental context. For molecules with end loops comprised of at least four T's CD spectra were virtually indistinguishable. However, these spectra differed considerably from the CD spectrum of the T2-looped molecule. The CD spectrum of the dumbbell with T3 end loops displayed features common to the dumbbells with larger loops and T2 end loops. Thermodynamic evidence that the terminal G.C base pairs (bps) nucleating the T2 end loops were intact was obtained from a comparison of the melting temperature of this molecule with that of a DNA dumbbell containing the 14 central bps of the set 1 duplex sequence linked instead by end loops comprised of the four base sequence, C-T-T-C. The tm of this latter molecule was determined to be 9 degrees C less than that of the former dumbbell assumed to contain a 16-bp stem and T2 end loops.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Composition↗

PubMed2000

Analysis of conformational changes at the unique loop adjacent to the ATP binding site of smooth muscle myosin using a fluorescent probe.

Recent crystallographic studies have shown that smooth muscle myosin has three highly conserved unique loops, loop B (320-327), loop M (687-699), and loop N (125-134), similar to other myosins, skeletal muscle and dictyostelium myosins. We previously demonstrated that the effect of actin is mediated by a conformational change in one of the loops, loop M comprising amino acids 677 to 689 of skeletal muscle myosin [Maruta and Homma (1998) J. Biochem. 124, 528-533]. In the present study, in order to clarify the role of these smooth muscle myosin loops in energy transduction, we specifically labeled the loops with a fluorescent photoreactive ADP analogue, 3'-O-(N-methylanthraniloyl)-8-azido-ADP (Mant-8-N(3)-ADP), and then measured the fluorescent polarization. When Mant-8-N(3)-ADP was trapped by aluminium fluoride or vanadate into the ATPase site, Mant-8-N(3)-ADP was covalently incorporated into loop N (125-134). In contrast, Mant-8-N(3)-ADP trapped by beryllium fluoride was covalently incorporated into both loop M (687-699) and loop N (125-134) at an almost equimolar ratio. Actin binding to smooth muscle myosin S1 (SMO-S1) labeled at only loop N (125-134) increased the polarization due to the viscosity of actin. In contrast, S1 labeled at both loops N and M showed a much smaller increase in polarization. Our results indicate that the probe at loop M (687-699) of smooth muscle myosin moved to a less hindered region, suggesting that actin binding induces conformational changes at loop M (687-699) similar to those of the corresponding loop (677-689) in skeletal muscle myosin, as previously demonstrated in our laboratory.

Adenosine Diphosphate↗

PubMed2004

Moment to force ratio characteristics of three Japanese NiTi and TMA dosing loops.

OBJECTIVES: This in vitro study investigated the loads ("forces"), moments and moment:force ratios (M:F) generated during activation and deactivation of three closing loop designs constructed from two different orthodontic wire alloys. METHOD: The forces and moments of non-preactivated vertical U-Loops, symmetrical T-Loops, and asymmetrical T-Loops (X-Loops) made from titanium molybdenum alloy (TMA) and Japanese nickel titanium alloy (NiTi) were measured at 35.6 degrees C +/- 0.5 degrees C. The M:F ratios generated during activation and deactivation were calculated for each loop. Analysis of covariance was used to identify statistical differences between loop material and design. RESULTS: The forces, moments and M:F ratios produced by the NiTi closing loops were significantly less than those from the TMA loops. NiTi T-Loops produced a relatively constant force during activation compared to the same design in TMA. The TMA X-loop produced a maximum M:F ratio of 9.4:1. No closing loop produced the optimum M:F ratio required for translational en masse tooth movement. CONCLUSIONS: Optimum M:F ratios for translation are not possible with non-preactivated vertical U-Loops, symmetrical T-Loops, and asymmetrical T-Loops (X-Loops) made of NiTi and TMA alloys.

Dental Alloys↗

PubMed2003

The nature of the turn in omega loops of proteins.

An analysis of Omega loops in a nonredundant set of protein structures from the Protein Data Bank has been carried out to determine the nature of the "turn elements" present. Because Omega loops essentially reverse their direction in three-dimensional space, this analysis was made with respect to four turn elements identified as (1) Gly; (2) Pro; (3) a residue with alpha-helical phi,psi angles, termed a helical residue; and (4) a cis peptide. A set of 1079 Omega loops from a set of 680 proteins were used for the analysis. Apart from other criteria that define Omega loops, the selection of an Omega loop from a cluster of loops is based on an exposure index. In this study, analyses have been made with two sets of data: (1) Omega loops arising from a minimum exposure index indicative of a less exposed loop (xmin set) and (2) Omega loops with a maximum exposure index indicative of a relatively exposed loop (xmax set). Overall residue preferences and positional preferences have been examined. Positions of the turn elements for Omega loops of varying length have also been studied. Specific positional preferences are observed for particular turn elements with regard to the length of Omega loops. Analysis in terms of the turn elements can provide guidelines for modeling of loops in proteins. Apart from Pro, which has the natural tendency to form cis peptide bonds, a higher occurrence of non-Pro cis peptide bonds is observed. Torsion angles in Omega loops also indicate the occurrence of a large number of residues with helical phi,psi angles, necessary for the turn in the loop structures.

Algorithms↗

PubMed2004

Factors affecting thermodynamic stabilities of RNA 3 x 3 internal loops.

Internal loops in RNA are important for folding and function. The 3 x 3 nucleotide internal loops are the smallest size symmetric loops with a potential noncanonical base pair (middle pair) flanked on both sides by a noncanonical base pair (loop-terminal pair). Thermodynamic and structural insights acquired for 3 x 3 loops should improve approximations for stabilities of 3 x 3 and larger internal loops. Most natural 3 x 3 internal loops are purine rich, which is also true of other internal loops. A series of oligoribonucleotides containing different 3 x 3 internal loops were studied by UV melting and imino proton NMR. Both loop-terminal and middle pairs contribute to the thermodynamic stabilities of 3 x 3 loops. Extra stabilization of -1.2 kcal/mol was found for a GA middle pair when flanked by at least one non-pyrimidine-pyrimidine loop-terminal pair. A penalty of approximately 1 kcal/mol was found for loops with a single loop-terminal GA pair that has a U 3' to the G of the GA pair. A revised model for predicting stabilities of 3 x 3 loops is derived by multiple linear regression.

Base Pairing↗

PubMed1997

Temperature dependence of backbone dynamics in loops of human mitochondrial heat shock protein 10.

A highly flexible, yet conserved polypeptide loop of Hsp10 mediates binding to Hsp60 in the course of chaperonin-dependent protein folding. Previous transferred nuclear Overhauser effect (trNOE) studies with peptides based on the mobile loop of the Escherichiacoli and bacteriophage T4 Hsp10s suggested that the mobile loop adopts a characteristic hairpin turn upon binding to the E. coli Hsp60 GroEL. In this paper, we identify the sequence and characterize the nascent structure and dynamics of the 18-residue mobile loop in the 15N-enriched human Hsp10. We also identify four residues of another flexible loop, the roof beta hairpin. The mobile loop and/or roof beta hairpin of several subunits are absent from the X-ray crystal structure of human Hsp10. NMR data suggest that the mobile loop of Hsp10 preferentially samples a hairpin conformation despite the fact that the backbone motion resembles that of a disordered polypeptide. Analysis of backbone dynamics by measurement of 15N relaxation times, T1 and T2, and the 1H-15N nuclear Overhauser effect (1H-15N NOE) indicates that motion is greatest near the center of the loop. Inversion of the temperature dependence of the T1 near the center of the loop marks a transition to motion with a dominant time scale of less than 3 ns. Analysis of the relaxation data by spectral density mapping shows that subnanosecond motion increases uniformly along the loop at elevated temperatures, whereas nanosecond motion increases near the ends of the loop and decreases near the center of the mobile loop. The transition to dominance by fast motion in the center of the loop occurs at a distance from the well-structured part of Hsp10 that is equal to the persistence length of an unstructured polypeptide. Simulation of the spectral density function for the 15N resonance and its temperature dependence using the Lipari-Szabo formalism suggests that the dominant time scales of loop motion range from 0.6 to 18 ns. For comparison, the time scale for molecular rotation of the 70 kDa Hsp10 heptamer is estimated to be 37 ns. Complex behavior of the T2 relaxation time indicates that motion also occurs on longer time scales. All of the modes of loop motion are likely to have an impact on Hsp10/Hsp60 interaction and therefore affect Hsp10/Hsp60 function as a chaperonin.

Amino Acid Sequence↗

PubMed2002

Complex role of the beta 2-beta 3 loop in the interaction of U1A with U1 hairpin II RNA.

RNA recognition motifs (RRMs) are characterized by highly conserved regions located centrally on a beta-sheet, which forms the RNA binding surface. Variable flanking regions, such as the loop connecting beta-strands 2 and 3, are thought to be important in determining the RNA-binding specificities of individual RRMs. The N-terminal RRM of the spliceosomal U1A protein mediates binding to an RNA hairpin (U1hpII) in the U1 small nuclear RNA. In this complex, the beta(2)-beta(3) loop protrudes through the 10-nucleotide RNA loop. Shortening of the RNA loop strongly perturbs binding, suggesting that an optimal "fit" of the beta(2)-beta(3) loop into the RNA loop is an important factor in complexation. To understand this interaction further, we mutated or deleted loop residues Lys(50) and Met(51), which protrude centrally into the RNA loop but do not make any direct contacts to the bases. Using BIACORE, we analyzed the ability of these U1A mutants to bind to wild type RNAs, or RNAs with shortened loops. Alanine replacement mutations only modestly affected binding to wild type U1hpII. Interestingly, simultaneous replacement of Lys(50) and Met(51) with alanine appeared to alleviate the loss of binding caused by shortening of the RNA loop. Deletion of Lys(50) or Met(51) caused a dramatic loss in stability of the U1A.U1hpII complex. However, deletion of both residues simultaneously was much less deleterious. Simulated annealing molecular dynamics analyses suggest this is due to the ability of this mutant to rearrange flanking amino acids to substitute for the two deleted residues. The double deletion mutant also exhibited substantially reduced negative effects of RNA loop shortening, suggesting the rearranged loop is better able to accommodate a short RNA loop. Our results indicate that one of the roles of the beta(2)-beta(3) loop is to provide a steric fit into the RNA loop, thereby stabilizing the RNA.protein complex.

Alanine↗

PubMed2001

Efficient repair of large DNA loops in Saccharomyces cerevisiae.

Small looped mispairs are efficiently corrected by mismatch repair. The situation with larger loops is less clear. Repair activity on large loops has been reported as anywhere from very low to quite efficient. There is also uncertainty about how many loop repair activities exist and whether any are conserved. To help address these issues, we studied large loop repair in Saccharomyces cerevisiae using in vivo and in vitro assays. Transformation of heteroduplexes containing 1, 16 or 38 nt loops led to >90% repair for all three substrates. Repair of the 38 base loop occurred independently of mutations in key genes for mismatch repair (MR) and nucleotide excision repair (NER), unlike other reported loop repair functions in yeast. Correction of the 16 base loop was mostly independent of MR, indicating that large loop repair predominates for this size heterology. Similarities between mammalian and yeast large loop repair were suggested by the inhibitory effects of loop secondary structure and by the role of defined nicks on the relative proportions of loop removal and loop retention products. These observations indicate a robust large loop repair pathway in yeast, distinct from MR and NER, and conserved in mammals.

Base Pair Mismatch↗

PubMed2000

Accuracy and precision of quantitative calibrated loops in transfer of bronchoalveolar lavage fluid.

Quantitative cultures of bronchoalveolar lavage (BAL) fluid are important in the diagnosis of ventilator-associated pneumonia, and calibrated loops are commonly used to set up these cultures. In this study, the performances of calibrated 0.010- and 0.001-ml loops in the transfer of BAL fluid were determined. Five loops of one lot from seven manufacturers were tested. Calibrations were performed by the gravimetric method (0.010-ml loops) and the colorimetric method (0.001-ml loops). Most of the 0.010-ml loops displayed a precision that was less than 10%, but six of them showed very poor accuracies as they transferred a deficiency (nichrome loops) or an excess (disposable loops) of BAL fluid that exceeded +/-10%. The mean maximum and minimum BAL fluid volumes delivered by the 0.010-ml loops differed by a factor 3. The 0.001-ml loops displayed acceptable precision. Five of them showed inaccuracies of </=+/-10%, and mean maximum and minimum BAL fluid volumes had a range of a factor of 2. For all loops, the volumes of BAL fluid sampled were larger than the volumes of reagent-grade water sampled. Results of the colony counting experiments confirmed these findings and revealed a high intra-assay variability for the 0.001-ml loops. We conclude that, when BAL fluid samples are cultured with calibrated loops, (i) proper verification of the calibration of these loops is mandatory, (ii) calibrations should be performed with BAL fluid as the test solution, and (iii) borderline quantitative culture results should be interpreted with knowledge of the inaccuracy values of these loops.

Bronchoalveolar Lavage Fluid↗

PubMed1999

Amphotropic murine leukemia virus entry is determined by specific combinations of residues from receptor loops 2 and 4.

Pit2 is the human receptor for amphotropic murine leukemia virus (A-MuLV); the related human protein Pit1 does not support A-MuLV entry. Interestingly, chimeric proteins in which either the N-terminal or the C-terminal part of Pit2 was replaced by the Pit1 sequence all retained A-MuLV receptor function. A possible interpretation of these observations is that Pit1 harbors sequences which can specify A-MuLV receptor function when presented in a protein context other than Pit1, e.g., in Pit1-Pit2 hybrids. We reasoned that such Pit1 sequences might be identified if presented in the Neurospora crassa protein Pho-4. This protein is distantly related to Pit1 and Pit2, predicted to have a similar membrane topology with five extracellular loops, and does not support A-MuLV entry. We show here that introduction of the Pit1-specific loop 2 sequence conferred A-MuLV receptor function upon Pho-4. Therefore, we conclude that (i) a functional A-MuLV receptor can be constructed by combining sequences from two proteins each lacking A-MuLV receptor function and that (ii) a Pit1 sequence can specify A-MuLV receptor function when presented in another protein context than that provided by Pit1 itself. Previous results indicated a role of loop 4 residues in A-MuLV entry, and the presence of a Pit2-specific loop 4 sequence was found here to confer A-MuLV receptor function upon Pho-4. Moreover, the introduction of a Pit1-specific loop 4 sequence, but not of a Pit2-specific loop 4 sequence, abolished the A-MuLV receptor function of a Pho-4 chimera harboring the Pit1-specific loop 2 sequence. Together, these data suggest that residues in both loop 2 and loop 4 play a role in A-MuLV receptor function. A-MuLV is, however, not dependent on the specific Pit2 loop 2 and Pit2 loop 4 sequences for entry; rather, the role played by loops 2 and 4 in A-MuLV entry can be fulfilled by several different combinations of loop 2 and loop 4 sequences. We predict that the residues in loops 2 and 4, identified in this study as specifying A-MuLV receptor function, are to be found among those not conserved among Pho-4, Pit1, and Pit2.

Amino Acid Sequence↗

PubMed1975

Presence of displacement loops in the covalently closed circular chloroplast deoxyribonucleic acid from higher plants.

Chloroplast DNAs (ctDNA) from pea and corn plants were examined in the electron microscope for the presence of replicative intermediates. Pea and corn ctDNAs were each found to contain two displacement loops (D-loops). The D-loops were 820 (+/- 90) base pairs long in pea ctDNA and 860 (+/- 125) base pairs long in corn ctDNA. In each ctDNA, the two D-loops were located at positions that were 7100 +/- 240) base pairs apart. The displacing strands of the two D-loops were located on opposite strands of the parental DNA molecule and they were seen to expand toward each other. The D-loops in the ctDNA from pea and corn exhibited branch migration and thus were easily distinguished from the denatured regions that were also present in these closed circular ctDNAs. In addition, the positions of the D-loops were found to be distinct from the positions of the denaturation loops (Den-loops). The Den-loops were also shown to be located at AT-rich regions in these ctDNA molecules. D-loops and Den-loops were also found in the circular and catenated ctDNA oligomers from pea and corn plants. Mapping the positions of the D-loops relative to the positions of the Den-loops showed that the structure of the D-loop-containing region in the pea and corn ctDNAs has been conserved to a greater extent than the structure of the rest of the two ctDNA molecules.

Binding Sites↗

PubMed1992

Substitution of putative half-cystine residues in heparin-binding fibroblast growth factor receptors. Loss of binding activity in both two and three loop isoforms.

Alternate use of an exon coding for an 89-residue NH2 terminal immunoglobulin-like disulfide loop results in isoforms of the heparin-binding fibroblast growth factor receptor (FGF-R) with three (FGF-R alpha) and two (FGF-R beta) Ig-like loops in the extracellular domain. Both FGF-R alpha and FGF-R beta isoforms exhibit qualitatively similar ligand-binding activities. In this report, we show by site-directed mutagenesis and analysis of ligand-binding activity in transfected cells that substitution of a cysteine that potentially forms an intra-loop disulfide in either juxtamembrane Loop II or III disrupted maturation and formation of the ligand-binding site in both FGF-R alpha and FGF-R beta isoforms. Neither three loop FGF-R alpha constructions coding for intact Loops I and II adjacent to defective Loop III nor intact Loops I and III separated by defective Loop II exhibited ligand-binding activity. In addition, a two-loop molecule of tandem Loops I and III was inactive. The results suggest that single Loops I, II, or III of FGF-R are insufficient to form a ligand-binding site. Loop I does not form an independent ligand-binding site with either Loop II or III, but interacts with a common ligand-binding site formed by Loops II and III (Xu, J., Nakahara, M., Crabb, J. W., Shi, E., Matuo, Y., Fraser, M., Kan, M., Hou, J., and McKeehan, W. L. (1992) J. Biol. Chem. 267, 17792-17803, 1992).

Amino Acid Sequence↗

PubMedDate not supplied

[Positive predictive value and negative predictive value of spiral CT in the diagnosis of closed loop obstruction complicated by intestinal ischemia].

PURPOSE: To assess the positive predictive value and the negative predictive value of spiral CT in the diagnosis of "closed-loop" obstruction complicated by intestinal ischaemia. MATERIALS AND METHODS: Between January 1999 and January 2002, 120 patients with small intestine closed-loop obstruction who had undergone surgical laparotomy within 2-6 hours from abdominal and pelvic spiral CT were retrospectively reviewed. The CT scans were performed using 5-mm slice thickness, 1 s scan delay, 5 x 5-mm collimation and 1.5 pitch after administering i.v. iodinated contrast material (120 ml volume, 60 s scan delay, 2.5-3 ml/s rate) with an automatic injector. For the diagnosis of closed-loop obstruction the following CT findings were examined: identification of the transitional area, 'C', 'U' or 'beak' shape of the obstructed loops, radial distribution of the involved mesenteric vessels. For the diagnosis of loop ischaemia, the following findings were evaluated in addition to the CT signs described above: submucosal oedema, increased, reduced, or no enhancement of the loop walls, oedema of the mesenteric vessels, fluid within the loops or in the intraperitoneal spaces. RESULTS: 'U', 'C', 'beak' loop configuration with poor or no contrast enhancement of the obstructed loop walls, ascites, rotation and engorgement of mesenteric vessels enabled the diagnosis of 26 cases of closed-loop obstruction complicated by ischaemia, infarction. U- or C-shaped loops with radial distribution and/or rotation of mesenteric vessels towards the obstruction site suggested the correct diagnosis in 94 cases, but did not allow identification of ischaemia in 25 cases. The positive predictive value of spiral CT related to ischaemic loop complications was 100%; the negative predictive value was 73%. CONCLUSIONS: Spiral CT is a reliable imaging technique enabling the diagnosis of closed-loop obstruction with or without intestinal ischaemic complications. Any alterations detected in the trophic status of the loops or mesentery imply ischaemic complications requiring emergency surgery. On the contrary, if only CT signs of closed-loop obstruction are detected, the existence and/or development of ischaemia cannot be ruled out.

Adult↗

PubMed2001

Sequence codes for extended conformation: a neighbor-dependent sequence analysis of loops in proteins.

We performed an extensive sequence analysis on the loops of proteins. By dividing a loop databank derived from the Protein Data Bank into groups, we analyzed the chemical characteristics and the sequence preferences of loops of different lengths and loops connecting different secondary structures in proteins. We found that a large population of loops in our loop databank (94.4%) is either partially or completely surface-exposed. A majority of surface loops in proteins are hydrophilic, whereas the chemical characteristics of interior loops are relatively neutral according to Eisenberg's consensus hydrophobicity scale. As a first step in investigating the intrinsic sequence-structure relationship of loop sequences in proteins, we performed a neighbor-dependent sequence analysis that calculated the effect of the neighboring amino acid type on the loop propensity of residues in loops. This method enhances the statistical significance of residue propensity, thus allowing us to explore the positional preference of amino acids in loops. Our analysis yielded a series of amino acid dyads that showed high preference for loop conformation. The data presented in this study should prove useful for developing potential codes in recognizing loop sequences in proteins.

Amino Acids↗