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PubMed1996

Loop electrosurgical excision procedure for squamous intraepithelial lesions of the cervix: advantages and potential pitfalls.

OBJECTIVE: to evaluate the advantages and pitfalls of the loop electrosurgical excision procedure as applied to the diagnosis and treatment of cervical cancer precursors. METHODS: Loop electrosurgical excision procedure using local anesthesia and colposcopic guidance was performed in an outpatient clinical setting in 1189 consecutive patients referred for colposcopy for an abnormal Papanicolaou smear during a period of 4 years. RESULTS: Of the 1189 patients, 915 (77%) were managed in one sitting with the "see and treat" approach, and in 274 patients endocervical curettage and cervical biopsies preceded loop electrosurgical excision procedure. One hundred nineteen (10%) patients were lost to follow-up. Twenty-one patients had either adenocarcinoma in situ (15) or microinvasive squamous cell carcinoma (six) in the loop electrosurgical excision procedure specimen, whereas the electroexcised specimens contained no lesional tissue in 166 (14%) patients. Cure (ie, disease-free at 6 months or longer) was observed in 92% of the 883 evaluable patients after a single treatment and 95% after a repeat loop electrosurgical excision procedure. High-grade squamous intraepithelial lesion was successfully treated with loop electrosurgical excision procedure in 287 (93%) of 309 patients. Complications, mainly intra- and postoperative bleeding, occurred in 7% of the patients. In most loop electrosurgical excision procedure-negative cases, the referral cytologic diagnosis or colposcopy and/or history were false-positive on review, or the biopsies performed before loop electrosurgical excision procedure removed smaller areas of abnormal tissue. CONCLUSION: Loop electrosurgical excision procedure using the see and treat approach should be limited to cytologically and colposcopically unequivocal intraepithelial lesions, and depth of excision should be controlled by colposcopy using loop electrodes of appropriate size. In doubtful cases, particularly in the young patient, disease should be ascertained by expert histology and colposcopy before definite therapy. Loop electroexcision represents an attractive means of diagnosing and treating cervical cancer precursors.

Adenocarcinoma↗
PubMed1996

Effect of modification of all loop regions in the alpha- and beta-subunits of human choriogonadotropin on its signal transduction activity.

Human choriogonadotropin (hCG), according to its three dimensional structure as determined by X-ray diffraction, has three beta-hairpin loops each in the alpha and beta subunit designated as alpha 1, alpha 2 alpha 3 and beta 1 beta 2 and beta 3, respectively. Since similar beta-hairpin loops in NGF and TNF beta have been implicated in their direct interaction with the receptor, it prompted the present investigation to determine the role of such loops in receptor binding and post-receptor signaling events in hCG. Based on the three dimensional structure of hCG, radical mutations were introduced in the alpha loops by replacing hydrophobic alpha 18Phe and alpha 74Phe by hydrophilic Thr residues in the alpha 1 and alpha 3 loops, respectively, and positively charged alpha 45Lys by negatively charged Asp in the helical segment in the alpha 2 loop. The beta loops were mutated by replacement of the beta 1, beta 2 and beta 3 sequences with the corresponding hFSH sequences. These replacements included beta 22Gly, beta 24Pro and beta 25Val with Glu, Arg and Phe in beta 1, 45Leu Gln Gly Val Leu Pro Ala Leu Pro53 with Tyr Lys Asn Pro Ala Arg Pro Leu Ile in beta 2 and 73Pro Arg Gly with Ala His His in the beta 3 loop. Six mutants, hCG alpha 1 beta, hCG alpha 2 beta and hCG alpha 3 beta and hCG alpha beta 1, hCG alpha beta 2 and h CG alpha beta 3, were obtained by co-infection of the insect High-Five cells with baculovirus containing mutant alpha or beta cDNAs and that containing complimentary wild type beta or alpha cDNAs. The mutants were almost completely secreted in the culture medium and were over expressed at levels ranging between 4.5 to 29 micrograms/ml indicating that mutations had no effect on the secretion or subunit assembly of hCG. In order to remove any contaminating beta-subunit, the culture medium was passed through a column of an hCG beta-specific monoclonal antibody, B158. The receptor binding activity of the mutant hCG alpha 1 beta, in which alpha 18Phe was replaced with Thr, increased almost 200% relative to rehCG. Similarly, increase in the cAMP and progesterone stimulation by the mutant ranged between 150 to 200%. This increase is believed to be due to a short range conformational change in the mutant as a result of the mutation rather than direct involvement of alpha 18Phe in the receptor binding. The evidence in support of this was derived from the fact that the affinity or interaction between the two subunits was impaired as indicated by the first order rate constant of hCG alpha 1 beta (km = 4.1 x 10(-2) min-1) at pH 3.0 at 23 degrees C which is one order of magnitude greater relative to rehCG (kw = 4.6 x 10(-3) min-1). All other mutations had no effect on the receptor binding or signal transduction of hCG indicating that, unlike NGF or TNF beta, beta-hairpin loops in hCG were not directly involved in receptor binding or post-receptor signaling events. However, since the mutation in the alpha 1 loop affects the receptor binding site, its presence in the vicinity of the alpha 1 loop is highly likely.

Baculoviridae↗
PubMed2004

The nasal loop provides an alternative to percutaneous endoscopic gastrostomy in high-risk dysphagic stroke patients.

INTRODUCTION: In the management of dysphagic stroke patients, percutaneous endoscopic gastrostomies (PEGs) are frequently sited early due to the failure of nasogastric tube (NGT) feeding, with NGTs becoming displaced in over 58% of cases. PEG insertion is a procedure with significant mortality and morbidity. We adapted a novel technique of securing NGTs (a nasal loop) which is non-invasive, allows successful NG feeding and may avoid the need for PEG placement. AIMS: To show that nasal loops result in improved delivery of enteral nutrition. To compare the outcome and complication rate of nasal loop fed patients with those undergoing PEG feeding. METHODS: A 6 month prospective audit of dysphagic stroke patients who were referred for PEG. All patients who were referred with failed NG feeding within 28 days of presentation were offered a nasal loop. Patients who were 28 days post-stroke had a PEG placed if appropriate. The daily feed intake was monitored before and after nasal loop placement. Complication rates and patient outcomes were documented at 2 week and 3 month follow-up. RESULTS: Nasal loop group: 14 patients had a nasal loop for a median of 15 days. The median daily feed provided was 0% before nasal loop and 100% after. Four patients went on to recover normal swallowing, 4 patients died and 6 later proceeded to PEG. PEG group: Seven patients proceeded direct to PEG, 1 died and 6 were alive and PEG fed at 3 months. There were 6 complications from PEG insertion. No patients recovered normal swallowing. CONCLUSIONS: Nasal loops are safe, well tolerated, and effective at delivering full enteral nutrition. Nasal loops allow time for patients who may recover normal swallowing to do so, and thus avoid a PEG. Nasal loops avoid unnecessary PEG insertion in those with a poor prognosis who will not ultimately survive their initial stroke.

Aged↗
PubMed2003

A prospective randomized comparison of loop recorders versus Holter monitors in patients with syncope or presyncope.

BACKGROUND: The initial management of syncope or presyncope typically involves short-term cardiac monitoring using a Holter monitor. A loop recorder is used to extend the period of monitoring to increase the probability of obtaining a symptom-rhythm correlation. Loop recorders and Holter monitors are both used commonly, but their utility has not been compared prospectively. METHODS: Patients with syncope or presyncope referred for a Holter monitor or a loop recorder were assigned randomly to receive either a 48-hour Holter monitor or a loop recorder for 1 month as an initial diagnostic strategy. If the initial strategy was unsuccessful, patients were offered crossover to the alternate strategy. RESULTS: One hundred patients (mean [+/- SD] age, 56 +/- 20 years; 44 women) with syncope (n = 21), presyncope (n = 29), or both (n = 50) were enrolled. Of the 49 patients assigned randomly to an external loop recorder first, 31 (63%) had an arrhythmia identified or excluded, versus 12 (24%) of 51 patients assigned to a Holter monitor initially (P <0.0001). Arrhythmia was identified as a cause of syncope in 1 patient with a loop recorder, compared with no patients with a Holter monitor (P = 0.31). Of the 29 patients with negative results with Holter monitoring who went on to receive a loop recorder, 13 (45%) had arrhythmia excluded, compared with none of the 4 patients who crossed over to receive a Holter monitor. The overall probability of obtaining a symptom-rhythm correlation was 56% (44/78) for loop recorders versus 22% (12/55) for Holter monitors (P <0.0001). Despite patient education and test transmissions, 13 (23%) of 57 patients who had recurrence of their symptoms failed to activate their loop recorder properly. CONCLUSION: Loop recorders have a much higher diagnostic yield for patients with syncope or presyncope as compared with Holter monitors. The utility of loop recorders is limited by some patients' inability to operate them correctly.

Electrocardiography, Ambulatory↗
PubMed2001

A semiflexible polymer model applied to loop formation in DNA hairpins.

A statistical mechanical "zipper" model is applied to describe the equilibrium melting of short DNA hairpins with poly(dT) loops ranging from 4 to 12 bases in the loop. The free energy of loop formation is expressed in terms of the persistence length of the chain. This method provides a new measurement of the persistence length of single-stranded DNA, which is found to be approximately 1.4 nm for poly(dT) strands in 100 mM NaCl. The free energy of the hairpin relative to the random coil state is found to scale with the loop size with an apparent exponent of > or = 7, much larger than the exponent of approximately 1.5-1.8 expected from considerations of loop entropy alone. This result indicates a strong dependence of the excess stability of the hairpins, from stacking interactions of the bases within the loop, on the size of the loop. We interpret this excess stability as arising from favorable hydrophobic interactions among the bases in tight loops and which diminish as the loops get larger. Free energy profiles along a generalized reaction coordinate are calculated from the equilibrium zipper model. The transition state for hairpin formation is identified as an ensemble of looped conformations with one basepair closing the loop, and with a lower enthalpy than the random coil state. The equilibrium model predicts apparent activation energy of approximately -11 kcal/mol for the hairpin closing step, in remarkable agreement with the value obtained from kinetics measurements.

Base Composition↗
PubMed2002

Hsc70-interacting HPD loop of the J domain of polyomavirus T antigens fluctuates in ps to ns and micros to ms.

The backbone dynamics of the J domain from polyomavirus T antigens have been investigated using 15N NMR relaxation and molecular dynamics simulation. Model-free relaxation analysis revealed picosecond to nanosecond motions in the N terminus, the I-II loop, the C-terminal end of helix II through the HPD loop to the beginning of helix III, and the C-terminal end of helix III to the C terminus. The backbone dynamics of the HPD loop and termini are dominated by motions with moderately large amplitudes and correlation times of the order of a nanosecond or longer. Conformational exchange on the microsecond to millisecond timescale was identified in the HPD loop, the N and C termini, and the I-II loop. A 9.7ns MD trajectory manifested concerted swings of the HPD loop. Transitions between major and minor conformations of the HPD loop featured distinct patterns of change in backbone dihedral angles and hydrogen bonds. Fraying of the C-terminal end of helix II and the N-terminal end of helix III correlated with displacements of the HPD loop. Correlation of crankshaft motions of Gly46 and Gly47 with the collective motions of the HPD loop suggested an important role of the two glycine residues in the mobility of the loop. Fluctuations of the HPD loop correlated with relative reorientation of side-chains of Lys35 and Asp44 that interact with Hsc70.

Antigens, Polyomavirus Transforming↗
PubMed2003

Activation loop phosphorylation and catalysis in protein kinases: is there functional evidence for the autoinhibitor model?

Many protein kinases are activated strongly by the phosphorylation of a polypeptide region (activation loop) that lies outside the active-site cleft. Analysis of the X-ray crystallographic structures of the insulin receptor with the activation loop in the phosphorylated and dephosphorylated forms offers a testable model for the mechanism of activity regulation by the loop. In this model, the dephosphorylated activation loop can act as an autoinhibitor by blocking substrate access to the active site. Phosphorylation of the loop could then release the autoinhibitor from the active site, allowing substrate binding and catalysis. While this model has been widely invoked, it was not clear if solution studies would support an autoinhibitory model for kinase regulation, in general. We review the results of solution studies on six protein kinases that test the role of the activation loop in controlling active-site access. While loop phosphorylation enhances substrate binding in two cases, four protein kinases display little or no effect on substrate dissociation constants. By comparison, phosphorylation increases catalysis by 2-4 orders of magnitude in all cases. These findings can be used to place the phosphorylatable activation loops into two broad, functional subcategories. (i) Gated activation loops exhibit bifunctional properties restricting substrate access and controlling catalysis. (ii) Nongated activation loops allow free movement of the substrate in and out of the active site irrespective of phosphorylation state but potently modulate the phosphoryl transfer step. Thus, while activation loop phosphorylation greatly modulates catalytic potential, it does not necessarily affect substrate binding, as once widely believed.

Catalysis↗
PubMed1997

Rapid loop dynamics of Yersinia protein tyrosine phosphatases.

The Yersinia protein tyrosine phosphatases (PTPase) contain a single and invariant tryptophan (W354) located at one of the hinge positions of the flexible loop (WpD loop), which is essential for catalysis. The wild-type Yersinia PTPase and an active site mutant in which the esential Cys 403 has been replaced by serine (C403S) have been examined using both time-resolved fluorescence anisotropy and steady-state UV resonance Raman (UVRR) spectroscopies. Both enzymes were examined with and without the bound inhibitor arsenate. The UVRR spectra indicate that in solution the ligand-free, wild-type PTPase exists as an equilibrium mixture of two tryptophan rotamer structures with chi2,1 dihedral angles of -4 degrees and -90 degrees. The two rotamers have been attributed to the presence of both "closed" and "open" WpD loop conformers of the ligand-free enzyme. Conversely, the UVRR spectra of the arsenate-ligated, wild-type PTPase and of ligand-free and arsenate-ligated C403S PTPase contain a single W3 band which is correlated to the -4 degrees rotamer of W354, indicating a predominance of the closed WpD loop conformer. The tryptophan fluorescence anisotropy decay measurements of the ligand-bound, wild-type Yersinia PTPase and of both ligation states of the C403S PTPase reveal a single correlation time of 30-48 ns due to the rotational motion of the protein, while the ligand-free, wild-type PTPase is found to have two correlation times of 31 and 3.8 ns. The 3.8 ns correlation time of the ligand-free enzyme is attributed to the hinged movement of the WpD loop which contains W354. These results indicate that under physiological conditions, the nonligated, wild-type Yersinia PTPase alternates between an open WpD loop and a closed loop form with a rate constant of approximately 2.6 x 10(8) s(-1). We conclude that the rate of WpD loop closure of the wild-type Yersinia PTPase is thus independent of the presence of ligand, whereas in the presence of ligand the rate of opening is dramatically reduced resulting in a closed conformation on ligand binding. In contrast, the ligand-free and ligated C403S PTPase remain in the loop closed configuration over the time course of our dynamic measurements. The lack of WpD loop motion in the C403S PTPase is believed to be due to either a loss of repulsive potential between the anionic thiolate and Asp 356 of the WpD loop and/or the formation of a hydrogen bond or water bridged hydrogen bond between Ser 403 and Asp 356.

Protein Conformation↗
PubMed1998

Sequence and context dependence of EF-hand loop dynamics. An 15N relaxation study of a calcium-binding site mutant of calbindin D9k.

The influence of amino acid sequence and structural context on the backbone dynamics of EF-hand calcium-binding loops was investigated using 15N spin relaxation measurements on the calcium-free state of the calbindin D9k mutant (A14D+A15Delta+P20Delta+N21G+P43M), in which the N-terminal pseudo-EF-hand loop, characteristic of S100 proteins, was engineered so as to conform with the C-terminal consensus EF-hand loop. The results were compared to a previous study of the apo state of the wild-type-like P43G calbindin D9k mutant. In the helical regions, the agreement with the P43G data is excellent, indicating that the structure and dynamics of the protein core are unaffected by the substitutions in the N-terminal loop. In the calcium-binding loops, the flexibility is drastically decreased compared to P43G, with the modified N-terminal loop showing a motional restriction comparable to that of the surrounding helixes. As in P43G, the motions in the C-terminal loop are less restricted than in the N-terminal loop. Differences in key hydrogen-bonding interactions correlate well with differences in dynamics and offer insights into the relationship between structure and dynamics of these EF-hand loops. It appears that the entire N-terminal EF-hand is built to form a rigid structure that allows calcium binding with only minor rearrangements and that the structural and dynamical properties of the entire EF-hand--rather than the loop sequence per se--is the major determinant of loop flexibility in this system.

Amino Acid Sequence↗
PubMed2005

Effect of proline and glycine residues on dynamics and barriers of loop formation in polypeptide chains.

Glycine and proline residues are frequently found in turn and loop structures of proteins and are believed to play an important role during chain compaction early in folding. We investigated their effect on the dynamics of intrachain loop formation in various unstructured polypeptide chains. Loop formation is significantly slower around trans prolyl peptide bonds and faster around glycine residues compared to any other amino acid. However, short loops are formed fastest around cis prolyl bonds with a time constant of 6 ns for end-to-end contact formation in a four-residue loop. Formation of short loops encounters activation energies in the range of 15 to 30 kJ/mol. The altered dynamics around glycine and trans prolyl bonds can be mainly ascribed to their effects on the activation energy. The fast dynamics around cis prolyl bonds, in contrast, originate in a higher Arrhenius pre-exponential factor, which compensates for an increased activation energy for loop formation compared to trans isomers. All-atom simulations of proline-containing peptides indicate that the conformational space for cis prolyl isomers is largely restricted compared to trans isomers. This leads to decreased average end-to-end distances and to a smaller loss in conformational entropy upon loop formation in cis isomers. The results further show that glycine and proline residues only influence formation of short loops containing between 2 and 10 residues, which is the typical loop size in native proteins. Formation of larger loops is not affected by the presence of a single glycine or proline residue.

Computer Simulation↗
PubMed1996

Glycosylation of multiple extracytosolic loops in Band 3, a model polytopic membrane protein.

N-glycosylated sites in polytopic membrane proteins are usually localized to single extracytosolic (EC) loops containing more than 30 residues [Landolt-Marticorena and Reithmeier (1994) Biochem. J. 302, 253-260]. This may be due to a biosynthetic restriction whereby only a single loop of nascent polypeptide is available to the oligosaccharyl transferase in the lumen of the endoplasmic reticulum. To test this hypothesis, two types of N-glycosylation mutants were constructed using Band 3, a polytopic membrane protein that contains up to 14 transmembrane segments and a single endogenous site of N-glycosylation at Asn-642 in EC loop 4. In the first set of mutants, an additional N-glycosylation acceptor site (Asn-Xaa-Ser/Thr) was constructed by site-directed mutagenesis in EC loop 3, with or without retention of the endogenous site. In the second set of mutants, EC loop 4 was duplicated and inserted into EC loop 2, again with or without retention of the endogenous site. Cell-free translation experiments using reticulocyte lysates showed that microsomes were able to N-glycosylate multiple EC loops in these Band 3 mutants. The acceptor site in EC loop 3 was poorly N-glycosylated, probably due to the suboptimal size (25 residues) of this EC loop. The localization of N-glycosylation sites to single EC loops in multi-span membrane proteins is probably due to the absence of suitably positioned acceptor sites on multiple loops.

Amino Acid Sequence↗
PubMed1979

Structural and functional alterations in the mucosa of self-filling intestinal blind loops in rats.

1. Self-filling blind loops of jejunum were constructed in three groups of rats; in the first, blind loops were created without further manipulation; in the second the bile was diverted permanently into the lower ileum below the blind loop, whereas in a third neomycin was added to the drinking water throughout the experiment. Two weeks after the creation of the blind loops, they were used for structural and functional studies. 2. Morphometric and microdissection techniques demonstrated that the surface area of the individual villi of the mucosa of 'ordinary' blind loops had increased fourfold in comparison with corresponding control jejunum, whereas the increase was only twofold in rats with bile diversion or in the series treated with neomycin. There were proportional increases in crypt length and mitotoic activity of the crypts in all three series, which suggest that the alterations in the mucosa were due to hyperplasia in both villus and crypt compartments. 3. Sucrase, succinate dehydrogenase, alkaline phosphatase and non-specific esterase activities, determined biochemically or histochemically, were reduced in the mucosae of all blind loops, though the changes were most pronounced in the 'ordinary' blind loops. The accumulation of L-phenylalanine by mucosal slices in vitro was depressed, although the decrease was less marked in the series treated with neomycin. 4. These results suggest that both bacteria and deconjugated bile acids play a role in the development of the hyperplastic changes of the blind-loop mucosa, but that another factor might also be involved: as a possible candidate, stasis of the intestinal contents was considered. 5. To test this hypothesis, loops of rat colon were transposed into the jejunum. Above the transposed loop, the jejunal mucosa developed hyperplasia of both villus and crypt compartments, with a reduction in its ability to accumulate L-phenylalanine. It is argued that these changes, probably caused by stasis of the intestinal contents, are triggered off by the dilatation of the gut, which may also be implicated in the mucosal alterations in blind loops.

Alkaline Phosphatase↗
PubMed1999

In vivo and in vitro studies of major surface loop deletion mutants of the Escherichia coli K-12 maltoporin: contribution to maltose and maltooligosaccharide transport and binding.

The trimeric protein LamB of Escherichia coli K-12 (maltoporin) specifically facilitates the diffusion of maltose and maltooligosaccharides through the outer membrane. Each monomer consists of an 18-stranded antiparallel beta-barrel with nine surface loops (L1 to L9). The effects on transport and binding of the deletion of some of the surface loops or of combinations of several of them were studied in vivo and in vitro. In vivo, single-, DeltaL4, DeltaL5, DeltaL6, and double-loop deletions, DeltaL4 + DeltaL5 and DeltaL5 + DeltaL6, abolished maltoporin functions, but not the double deletion DeltaL4 + DeltaL6 and the triple deletion DeltaL4 + DeltaL5 + DeltaL6. While deletion of the central variable portion of loop L9 (DeltaL9v) affected maltoporin function only moderately, the combination of DeltaL9v with the double deletion of loops L4 and L6 (triple deletion DeltaL4 + DeltaL6 + DeltaL9v) strongly impaired maltoporin function and resulted in sensitivity to large hydrophilic antibiotics without change in channel size as measured in vitro. In vitro, the carbohydrate-binding properties of the different loop mutants were studied in titration experiments using the asymmetric and symmetric addition of the mutant porins and of the carbohydrates to one or both sides of the lipid bilayer membranes. The deletion of loop L9v alone (LamBDeltaL9v), of two loops L4 and L6 (LamBDeltaL4 + DeltaL6), of three loops L4, L5 and L6 (LamBDeltaL4 + DeltaL5 + DeltaL6) or of L4, L6 and L9v (LamBDeltaL4 + DeltaL6 + DeltaL9v) had relatively little influence on the carbohydrate-binding properties of the mutant channels, and they had approximately similar binding properties for carbohydrate addition to both sides compared with only one side. The deletion of one of the loops L4 (LamBDeltaL4) or L6 (LamBDeltaL6) resulted in an asymmetric carbohydrate binding. The in vivo and in vitro results, together with those of the purification across the starch column, suggest that maltooligosaccharides enter the LamB channel from the cell surface side with the non-reducing end in advance. The absence of some of the loops leads to obstruction of the channel from the outside, which results in a considerable difference in the on-rate of carbohydrate binding from the extracellular side compared with that from the periplasmic side.

Bacitracin↗
PubMed2002

Modelling of the III-IV loop, a domain involved in calcium channel Ca(v)2.1 inactivation, highlights a structural homology with the gamma subunit of G proteins.

We have modelled the conformation of the III-IV loop of the Ca(v)2.1 subunit of P/Q calcium channels, a loop that is implicated in fast voltage-dependent inactivation. Change in channel inactivation requires its direct interaction with the I-II loop. This interaction occurs with an affinity in the order of 70 nm. Intracellular injection of a 40-mer III-IV loop-derived peptide produces an increase in the rate of fast inactivation. This alteration in channel kinetic is also accompanied by a hyperpolarizing shift in the steady-state voltage-dependence of inactivation. None of these effects are observed in the presence of a beta subunit, suggesting the existence of a competitive mechanism of action between the beta subunit and the III-IV loop. Amino acid sequence comparison using BLAST reveals that the III-IV loop shares 53% identity with the gamma subunit of G proteins. Because of the pivotal contribution of the III-IV loop to inactivation, an atomic model of the III-IV loop was generated by both homology modelling and molecular mechanics calculations. Using the X-ray structures of the betagamma dimer of the heterotrimeric G-proteins as templates, the III-IV loop is predicted to contain a well-structured alpha-helix at the amino-terminus with both the N- and C-termini having the same orientation in the plane of the inner lipid bilayer. We provide a hypothetical working model in which we propose that the III-IV loop interacts with the I-II loop via its Gbetagamma binding domain.

Amino Acid Sequence↗
PubMed1995

Alternately spliced NH2-terminal immunoglobulin-like Loop I in the ectodomain of the fibroblast growth factor (FGF) receptor 1 lowers affinity for both heparin and FGF-1.

Alternate splicing of a single exon encoding an NH2-terminal immunoglobulin (Ig) disulfide loop in the ectodomain of the fibroblast growth factor receptor (FGFR) types 1 and 2 results in alpha and beta isoforms that exhibit 3- and 2-Ig loops, respectively. Previously we demonstrated that alternately spliced Loop I has no independent ligand binding activity but is sufficiently interactive with the ligand- and heparin-binding site formed by Loops II and III to lower affinity for the same fibroblast growth factor (FGF) ligand. Here we show that a lower affinity of FGFR1 alpha for heparin parallels the lower affinity for FGF-1. A mutant of FGFR1 alpha in which the sequence between Loops I and II was deleted exhibits high affinity for both FGF-1 and heparin and other properties of the FGFR1 beta isoform, which include resistance to degradation by trypsin and display of specific antibody epitopes. This suggests that the interloop sequence facilitates the interaction of Loop I with Loops II and III. Lack of expression of both exons coding for Loop I and the sequence between Loops I and II in the FGFR2 gene characterizes rat prostate tumor cells, which exhibit a loss of the low affinity class of FGF receptors. Although the exon coding for the sequence between Loops I and II is alternately spliced in the FGFR2 beta isoform, coordinate expression with the exon coding for Loop I results in the functional differences between the FGFR alpha and FGFR beta variants.

Alternative Splicing↗
PubMed1999

Correction of large mispaired DNA loops by extracts of Saccharomyces cerevisiae.

Single base mispairs and small loops are corrected by DNA mismatch repair, but little is known about the correction of large loops. In this paper, large loop repair was examined in nuclear extracts of yeast. Biochemical assays showed that repair activity occurred on loops of 16, 27, and 216 bases, whereas a G-T mispair and an 8-base loop were poorly corrected under these conditions. Two modes of loop repair were revealed by comparison of heteroduplexes that contained a site-specific nick or were covalently closed. A nick-stimulated repair mode directs correction to the discontinuous strand, regardless of which strand contains the loop. An alternative mode is nick-independent and preferentially removes the loop. Both outcomes of repair were largely eliminated when DNA replication was inhibited, suggesting a requirement for repair synthesis. Excision tracts of 100-200 nucleotides, spanning the position of the loop, were observed on each strand under conditions of limited DNA repair synthesis. Both repair modes were independent of the mismatch correction genes MSH2, MSH3, MLH1, and PMS1, as judged by activity in mutant extracts. Together the loop specificity and mutant results furnish evidence for a large loop repair pathway in yeast that is distinct from mismatch repair.

Base Pair Mismatch↗
PubMed2005

Loop 1 of transducer region in mammalian class I myosin, Myo1b, modulates actin affinity, ATPase activity, and nucleotide access.

Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myosin-II for ADP, motility and the V(max) of the actin-activated Mg2+-ATPase activity, possibly through P(i) release (2-8). To test the influence of loop 1 on the mammalian class I myosin, Myo1b, chimeric molecules in which (i) loop 1 of a truncated form of Myo1b, Myo1b1IQ, was replaced with either loop 1 from other myosins; (ii) loop 1 was replaced with glycine; or (iii) some amino acids in the loop were substituted with alanine and were expressed in baculovirus, and their interactions with actin and nucleotide were evaluated. The steady-state actin-activated ATPase activity; rate of ATP-induced dissociation of actin from Myo1b1IQ; rate of ADP release from actin-Myo1b1IQ; and the affinity of actin for Myo1b1IQ and Myo1b1IQ.ADP differed in the chimeras versus wild type, indicating that loop 1 has a much wider range of effects on the coupling between actin and nucleotide binding events than previously thought. In particular, the biphasic ATP-induced dissociation of actin from actin-Myo1b1IQ was significantly altered in the chimeras. This provided evidence that loop 1 contributes to the accessibility of the nucleotide pocket and is involved in the integration of information from the actin-, nucleotide-, gamma-P(i)-, and calmodulin-binding sites and predicts that loop 1 modulates the load dependence of the motor.

Actins↗
PubMed1997

The 5' stem-loop from human U4 snRNA adopts a novel conformation stabilized by G-C and G-U base pairs.

1H NMR and molecular modeling studies of the 5' stem-loop from human U4 snRNA were undertaken to determine the conformation of this stem-loop that is essential for spliceosome formation and pre-mRNA splicing. Sixteen of the 35 nucleotides of this stem-loop are in the loop region and inspection of the loop sequence revealed no decomposition into elements of secondary structure commonly found in other RNA stem-loops. An analysis of possible base pairing interactions for this stem-loop using the methods of Zuker revealed the lowest energy secondary structure for the 16 nucleotide loop consisted of four base pairs at the base of a non-canonical tetraloop (UUUA). This shorter stem-loop was joined to the nine base pair stem by two A residues on the 5' side and a single bulged A on the 3' side. Both stems also had bulged A residues. 1H NMR experiments performed on solutions of the 35 mer stem-loop, the stem region, and the loop region confirmed the 35 mer adopted this secondary structure in solution. A 3D molecular model of this structure consistent with the NMR data was generated to assist in visualization of this novel structure.

Base Composition↗