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PubMed2003

Host factor titration by chromosomal R-loops as a mechanism for runaway plasmid replication in transcription termination-defective mutants of Escherichia coli.

Two Escherichia coli genes, rnhA and recG, encode products that disrupt R-loops by hydrolysis and unwinding, respectively. It is known that the propensity for R-loop formation in vivo is increased during growth at 21 degrees C. We have identified several links between rnhA, recG, and R-loop-dependent plasmid replication on the one hand, and genes rho and nusG involved in factor-dependent transcription termination on the other. A novel nusG-G146D mutation phenocopied a rho-A243E mutation in conferring global deficiency in transcription termination, and both mutants were killed at 21 degrees C following overexpression of rnhA(+). Mutant combinations rnhA-nusG or recG-rho were synthetically lethal at 21 degrees C, with the former being suppressed by recG(+) overexpression. rho and nusG mutants were killed following transformation with plasmids such as pACYC184 or pUC19 (which have R-loop replication intermediates) even at 30 degrees C or 37 degrees C, and the lethality was correlated with greatly increased content of supercoiled monomer species of these and other co-resident R-loop-dependent plasmids. Plasmid-mediated lethality in the mutants was suppressed by overexpression of rnhA(+) or recG(+). Two additional categories of trans-acting suppressors of the plasmid-mediated lethality were identified whose primary effects were, respectively, a reduction in plasmid copy number even in the wild-type strain, and a restoration of the proficiency of in vivo transcription termination in the nusG and rho mutant strains. The former category of suppressors included rom(+), and mutations in rpoB(Q513L), pcnB, and polA, whereas the latter included a mutation in rho (R221C) and several non-null mutations (E74K, L26P, and delta64-137) in the gene encoding the nucleoid protein H-NS. We propose that an increased occurrence of chromosomal R-loops in the rho and nusG mutants leads to titration of a cyloplasmic host factor(s) that negatively modulates the stability of plasmid R-loop replication intermediates and consequently to runaway plasmid replication.

Bacterial Proteins↗
PubMed1990

Prediction of rho-independent Escherichia coli transcription terminators. A statistical analysis of their RNA stem-loop structures.

Escherichia coli rho-independent transcription terminators are characterized by an RNA structure having a G+C-rich stem-loop followed by a series of uridine residues, but they can be only partially predicted by the stability of this structure or by its primary sequence. A large number of such terminators have been identified or proposed in the literature, and we have constituted a list of them (148 found in 1021 x 10(3) base-pairs of E. coli DNA sequences) in order to analyze statistically the corresponding RNA hairpins. We show that the size of the loops presents a narrow distribution, that their sequences are not random, and that most loops are closed by a C.G base-pair. In particular, 55% of the loops are tetranucleotides and the most abundant loop sequences are UUCG and GAAA. These loops are abundant in prokaryotic and eukaryotic RNAs, and are known to enhance the stability of RNA hairpins. We propose that these tetraloops play an important role in the nucleation of the nascent RNA structures, as does also the presence of a C.G base-pair closing a hairpin loop. This analysis allows us to propose a model of formation of an RNA hairpin during the termination process and to construct an algorithm of prediction of the terminators in a given DNA sequence. For the E. coli sequences, it clearly distinguishes inter- from intracistronic terminator-like structures, and selects 141 of the 148 rho-independent terminators given in the literature, with a very low background. It also predicts with reasonable accuracy the in vitro termination efficiency of known rho-independent terminators, as well as predicting the existence of 35 as yet uncharacterized terminators.

Algorithms↗
PubMed1998

Evaluation of new resectoscope loop for transurethral resection of bladder tumors.

PURPOSE: We evaluate a new resectoscope loop for transurethral resection of bladder tumors. MATERIALS AND METHODS: Of 251 transurethral resections in 226 patients 111 were done with a conventional loop and 140 with the Olympus prototype model A2186 resectoscope loop. The quality of specimens provided for histological analysis was compared. RESULTS: Tissue orientation was preserved and cautery artifact was reduced with the new loop compared to the standard resectoscope loop. CONCLUSIONS: Compared to a conventional resectoscope loop, the new loop resulted in a better tumor resection and helped facilitate histological evaluation of the tissue specimens.

Endoscopes↗
PubMed2006

Detection and structural analysis of R-loops.

R-loops are structures where an RNA strand is base paired with one DNA strand of a DNA duplex, leaving the displaced DNA strand single-stranded. Stable R-loops exist in vivo at prokaryotic origins of replication, the mitochondrial origin of replication, and mammalian immunoglobulin (Ig) class switch regions in activated B lymphocytes. All of these R-loops arise upon generation of a G-rich RNA strand by an RNA polymerase upon transcription of a C-rich DNA template strand. These R-loops are of significant length. For example, the R-loop at the col E1 origin of replication appears to be about 140 bp. Our own lab has focused on class switch regions, where the R-loops can extend well over a kilobase in length. Here, methods are described for detection and analysis of R-loops in vitro and in vivo.

Animals↗
PubMed1988

Decentration of flexible loop posterior chamber intraocular lenses in a series of 222 postmortem eyes.

Two hundred twenty-two postmortem eyes containing posterior chamber intraocular lenses (IOLs) were analyzed for optic decentration in relationship to lens style, implant duration, and loop fixation site. Decentration values were not affected significantly by either lens style or implant duration. In 33.3% of specimens, both loops were situated within the lens capsular sac, 18.0% had both loops fixated in the ciliary sulcus, and in 48.7% one loop was fixated in the lens capsular sac and the opposite loop in the ciliary sulcus or zonular region. There was a statistically significant difference in the amount of decentration in the three fixation groups studied. Capsular fixation provides the best and most consistent centration compared with fixation of both loops in the ciliary sulcus or asymmetrical fixation with only one loop in the capsular sac.

Ciliary Body↗
PubMed2000

Basal ganglia and cerebellar loops: motor and cognitive circuits.

The traditional view that the basal ganglia and cerebellum are simply involved in the control of movement has been challenged in recent years. One of the pivotal reasons for this reappraisal has been new information about basal ganglia and cerebellar connections with the cerebral cortex. In essence, recent anatomical studies have revealed that these connections are organized into discrete circuits or 'loops'. Rather than serving as a means for widespread cortical areas to gain access to the motor system, these loops reciprocally interconnect a large and diverse set of cerebral cortical areas with the basal ganglia and cerebellum. The properties of neurons within the basal ganglia or cerebellar components of these circuits resembles the properties of neurons within the cortical areas subserved by these loops. For example, neuronal activity within basal ganglia and cerebellar loops with motor areas of the cerebral cortex is highly correlated with parameters of movement, while neuronal activity within basal ganglia and cerebellar loops with areas of the prefrontal cortex is more related to aspects of cognitive function. Thus, individual loops appear to be involved in distinct behavioral functions. Studies of basal ganglia and cerebellar pathology support this conclusion. Damage to the basal ganglia or cerebellar components of circuits with motor areas of cortex leads to motor symptoms, whereas damage of the subcortical components of circuits with non-motor areas of cortex causes higher-order deficits. In this report, we review some of the new anatomical, physiological and behavioral findings that have contributed to a reappraisal of function concerning the basal ganglia and cerebellar loops with the cerebral cortex.

Animals↗
PubMed1997

V3 loop core region serotyping of HIV-1 infected patients using the FHV epitope presenting system.

We have reported recently a new epitope presenting system based on the Flock House Virus (FHV) capsid protein. The HIV-1 V3 loop core sequence IGPGRAF was inserted in different sites of this carrier molecule. Immunoreactivity experiments and molecular modelling consistently showed that the most reactive recombinant protein displayed the IGPGRAF sequence in a conformation which is most similar to that of a V3 loop reference structure. The same insertion site was then used to display the V3 loop apex sequences of six different HIV-1 isolates. Sera from 32 HIV-1 infected patients were examined for their reactivity to our chimeric proteins and the results were compared with those obtained using synthetic V3 loop peptides. The data obtained were confirmed by nested PCR amplification and direct sequencing of the patient's V3 loops. The results showed that the V3 loop serotyping using the FHV hybrid proteins, was more specific than that obtained using synthetic peptides. This system will therefore be a useful tool for the correct evaluation of the immune response against different V3 loop core sequences.

Amino Acid Sequence↗
PubMed2000

Dynamics of a mobile loop at the active site of Escherichia coli asparaginase.

Asparaginase II from Escherichia coli is well-known member of the bacterial class II amidohydrolases. Enzymes of this family utilize a peculiar catalytic mechanism in which a pair of threonine residues play pivotal roles. Another common feature is a mobile surface loop that closes over the active site when the substrates is bound. We have studied the motion of the loop by stopped-flow experiments using the fluorescence of tryptophan residues as the spectroscopic probe. With wild-type enzyme the fluorescence of the only tryptophan, W66, was monitored. Here asparagine induced a rapid closure of the loop. The rate constants of the process (100-150 s(-1) at 4 degrees C) were considerably higher than those of the rate-limiting catalytic step. A more selective spectroscopic probe was generated by replacing W66 with tyrosine and Y25, a component of the loop, with tryptophan. In the resulting enzyme variant, k(cat) and the rate of loop movement were reduced by factors of 10(2) and >10(3), respectively, while substrate binding was unaffected. This indicates that the presence of tyrosine in position 25 is essential for both loop closure and catalysis. Numerical simulations of the observed transients are consistent with a model where loop closure is an absolute prerequisite for substrate turnover.

Asparaginase↗
PubMed1997

Effect of modification of the beta-hairpin and long loops simultaneously in both alpha- and beta-subunits on the function of human choriogonadotropin: part II.

According to the X-ray diffraction, human choriogonadotropin has four beta-hairpin and two long loops, equally distributed in each of the alpha and beta subunits. Radical mutations such as the replacement of alpha 18Phe and alpha 74Phe with Thr in the alpha1 and alpha3 loops respectively and the replacement of alpha 45Lys with Asp in the alpha2 loop in the alpha-subunit were introduced while the loop sequences in the beta-subunit were replaced with the corresponding sequences in hFSH beta. Nine different double mutants with simultaneous mutations in both the alpha and beta loops including hCG alpha1 beta1, hCG alpha1 beta2, hCG alpha1 beta3, hCG alpha2 beta1, hCG alpha2 beta2, hCG alpha2 beta3, hCG alpha3 beta1, hCG alpha3 beta2 and hCG alpha3 beta3 were partially purified from insect High-Five cells. As previously reported (Shao et al., 1996, Mol. Cell. Endocrinol. 122, 173-182), the mutation in the alpha1 loop in the mutant, hCG alpha1 beta, the mutants hCG alpha1 beta1 and hCG alpha1 beta3 caused 200%, increase in the receptor binding, cAMP and progesterone stimulation. The mutant, hCG alpha1 beta2 and all other mutants behaved like the recombinant hCG (rehCG) in the receptor binding and post-receptor signaling activities. The molecular cause for this increase is probably due to a conformational change in the heterodimers caused by the mutation in the alpha1 loop. This conclusion is based on the results of the dissociation studies of the mutants heterodimers which indicated a decreased affinity between the subunits. The first order rate constants for the dissociation of the mutants hCG alpha1 beta1, hCG alpha1 beta2 and hCG alpha beta3 were 3.7 x 10(-2) min(-1), 1.4 x 10(-2) min(-1) and 4.6 x 10(-2) min(-1) respectively, as compared with 4.6 x 10(-3) min(-1) for the rehCG. It seems from the data that alpha 18Phe is located in, or in proximity to the receptor binding site and probably plays a critical role in maintaining either directly or indirectly its conformational integrity.

Animals↗
PubMed2001

Introns resolve the conflict between base order-dependent stem-loop potential and the encoding of RNA or protein: further evidence from overlapping genes.

Many eukaryotic genes are split into exons and introns, the latter being removed post-transcriptionally so that only exon sequences appear in cytoplasmic RNAs. Since introns appear in both protein-encoding RNAs and non-protein-coding RNAs, they interrupt genetic information per se, not just protein-encoding information. A DNA sequence has the potential to carry more than one type of genetic information, but different types may conflict. Thus, it has been proposed that introns arose because sequences were unable to contain concomitantly complete information for the encoding both of stem-loops and of cytoplasmic products (protein and/or RNA). Stem-loop potential is held to be selectively advantageous since it promotes the recombination-dependent correction of genetic errors. Stem-loop potential, the best local measure of which is base order-dependent stem-loop potential, tends to be less in exons than in introns. This is particularly evident in genes evolving rapidly under positive Darwinian selection, where the protein-encoding function is dominant. Evidence is now presented that the rare regions where genes overlap also impose excessive encoding demands so that the concomitant coding of base order-dependent stem-loop potential is decreased. Our results are consistent with the hypothesis that sequences with high stem-loop potential arose in the early 'RNA world'. Ancestors of modern genes would have entered this world when sequences (exons) encoding cytoplasmic products, were interspersed with sequences (introns) encoding selectively advantageous stem-loops. Purine-loading pressure would also have favoured intron formation.

Animals↗
PubMed1991

Pressure-length loop area: its components analyzed during graded myocardial ischemia.

The changes in total pressure-length loop area were compared with changes in effective shortening area, systolic lengthening area and postsystolic shortening area (defined with respect to end-diastolic and end-systolic lengths) of the pressure-length loop during myocardial ischemia in seven anesthetized dogs instrumented for measurement of left ventricular pressure and regional segmental wall motion (sonomicrometry) in the minor axis of the apical region of the left ventricle. Ischemia was induced by gradual tightening of a micrometer-controlled snare around the left anterior descending coronary artery, which supplied the apical myocardium. Data were obtained at normal flow, after critical constriction (loss of pulsatile coronary flow), mild ischemia (ischemia 1: onset of regional dysfunction, i.e., postsystolic shortening and mild hypokinesia) and moderate ischemia (ischemia 2: marked hypokinesia). At each stage, acute afterloading was performed by partially occluding the descending thoracic aorta. The pressure-length loops were analyzed in terms of four areas: total loop area, effective shortening area, postsystolic shortening area and systolic lengthening area. Total loop area decreased only when marked hypokinesia was present (176 +/- 18.3 mm Hg x mm at ischemia 2 versus 245.1 +/- 26.9 mm Hg x mm at ischemia 1, p less than 0.05). However, effective shortening area (98.2 +/- 0.8% of total loop area at baseline; 93.8 +/- 2.4% at critical constriction; 76.3 +/- 7.2% at ischemia 1; 51.9 +/- 12.2% at ischemia 2) and postsystolic shortening area (1.8 +/- 0.8% of total loop area at baseline; 5.2 +/- 1.9% at critical constriction; 14.3 +/- 3/4% at ischemia 1; 23.8 +/- 5.1% at ischemia 2) changed significantly with each progressive stage of ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

Loop security as a determinant of tissue fixation security.

Secure arthroscopic repair of rotator cuff tears and Bankart lesions requires tight knots (knot security). Equally important, but usually overlooked, is the tightness of the suture loop (loop security). This study compared loop security in knots tied with No. 1 PDS suture using three different methods: (1) hand-tied, (2) single-hole standard knot pusher, and (3) cannulated double-diameter knot pusher. The results of this study show that the double-diameter knot pusher maintained tight suture loops that were equivalent in circumference to hand-tied loops and were significantly tighter than suture loops tied with a standard single-hole knot pusher. This study highlights the fact that loop security is equally important to knot security in tissue fixation.

Arthroscopy↗
PubMed1987

In vivo fracture of an extruded polymethylmethacrylate intraocular lens loop.

The inferior loop of a semiflexible, closed-loop anterior chamber intraocular lens in the eye of a 72-year-old female fractured at two sites. The loop was composed of extruded polymethylmethacrylate. The inferotemporal portion of the loop was first noted to be broken 25 months postoperatively, and the fractured nasal portion of the same loop was first observed 55 months after implantation. The patient denied any excessive rubbing of the eye or major trauma to the eye or head. Both fractures occurred near the loop-optic junction. The intermittent touch of the cornea by the broken loop caused endothelial damage and edema in the lower one third of the cornea. The two portions of the lens were removed and exchanged with another style of anterior chamber lens. The visual acuity at six weeks postexchange was 20/30.

Aged↗
PubMed1999

Defunctioning loop ileostomy: a prospective audit.

BACKGROUND: Defunctioning loop ileostomies are uncommonly used forms of fecal diversion. The aim of this study was to determine the morbidity associated with both construction and reversal of loop ileostomies. STUDY DESIGN: An analysis was performed of all patients who underwent loop ileostomy construction or reversal between 1990 and 1997, with data being collected prospectively. RESULTS: One-hundred-two patients, (43 male, 59 female) with a mean age of 38 years (range 13 to 81 years) had loop ileostomies constructed. Indications for ileostomy construction included inflammatory bowel disease (76 patients), ultralow anterior resection for carcinoma (16 patients), or miscellaneous reasons (10 patients). Nine patients (9%) had complications arising from ileostomy construction including 4 parastomal infections, 3 high output ileostomies, 1 small-bowel obstruction, and 1 ileostomy stenosis in the early postoperative period. Only the stricture required surgical intervention. All other complications improved with conservative management. Mean time to ileostomy reversal was 120 days. Three patients (4%) had complications associated with reversal. All of these complications required surgical intervention, 2 for small bowel obstruction, and 1 for small bowel perforation. Currently 84 patients have had their ileostomy reversed, and 12 patients have had their loop ileostomy converted to a permanent stoma for reasons not related to the loop ileostomy itself. CONCLUSIONS: Defunctioning loop ileostomy is associated with low morbidity. We recommend a defunctioning ileostomy as the procedure of choice for temporary fecal diversion.

Adolescent↗
PubMed2004

Loop ileostomy: modification of technique.

INTRODUCTION: A loop ileostomy is a suitable procedure for faecal diversion. A number of technical improvements and advancement in stoma management have made its creation a suitable alternative to a loop colostomy. We describe an alternative technique for securing a loop ileostomy and perform a retrospective review of this technique. PATIENTS & METHOD: 40 patients who had a loop ileostomy performed as part of an abdominal procedure were reviewed. The loop of ileum was secured to the stoma site with a novel 'suture bridge' technique. RESULTS: 32 patients had the stoma formed to protect a distal anastomosis, 6 to palliate bowel obstruction, 1 to control faecal incontinence and another for colonic Crohn's disease. There were no incidences of paralytic ileus, mechanical obstruction, prolapse, retraction or bleeding after the loop ileostomies were formed. Thirty patients had their ileostomies closed. In 27 patients this was performed by excising the muco-cutaneous edge and anterior closure. Three patients had their stomas resected and an end-to-end bowel anastomoses. Following closure there were two complications in separate patients--self-limiting paralytic ileus and small bowel obstruction at the site of the stomal closure that required a second operation. There were no incidences of anastomotic leaks or bleeding in patients who had their ileostomy closed. No mortalities were attributed to either stoma formation or closure. CONCLUSION: We have described a safe alternative technique for securing a loop ileostomy with negligible complications in construction and closure as demonstrated in our results.

Adult↗
PubMed2003

Functional roles for amino acids in active site loop II of a hypoxanthine phosphoribosyltransferase.

A flexible loop of amino acids (loop II) closes over the active site of hypoxanthine phosphoribosyltransferase (HPRT) as the enzyme approaches the transition state [Biochemistry 37 (1998) 17120]. Formerly, the deletion of much of loop II from the HPRT of Trypanosoma cruzi resulted in a 2-3 order of magnitude reduction in k(cat) values with relatively modest changes in the Michaelis constants for substrates [Biochim. Biophys. Acta 1537 (2001) 63-70]. However, the contributions of individual loop II residues to catalysis remained poorly understood or have been disputed. Herein, saturation mutagenesis was used to generate relatively random sets of mutations in the 12 residues of active site loop II in the HPRT from T. cruzi and steady-state kinetics was used to investigate reactions catalyzed by the mutants. The results of analyses of 18 different mutations in an evolutionarily invariant Ser-Tyr dipeptide are consistent with interactions, between main chain nitrogen atoms of these residues and the O1A atom of phosphoribosylpyrophosphate (PRPP) or pyrophosphate (PPi), being essential for efficient enzyme chemistry. The results of analyses of 55 mutations in the nine other amino acids in loop II are inconsistent with these residues participating directly in enzyme chemistry, but are consistent with several of their side chains influencing loop flexibility and folding, as well as the efficiency for nucleotide formation relative to pyrophosphorolysis.

Amino Acid Substitution↗
PubMed2001

ATP-dependent interaction of yeast U5 snRNA loop 1 with the 5' splice site.

Pre-messenger RNA splicing is a two-step process by which introns are removed and exons joined together. In yeast, the U5 snRNA loop 1 interacts with the 5' exon before the first step of splicing and with the 5' and 3' exons before the second step. In vitro studies revealed that yeast U5 loop 1 is not required for the first step of splicing but is essential for holding the 5' and 3' exons for ligation during the second step. It is critical, therefore, that loop 1 contacts the 5' exon before the first step of splicing to hold this exon following cleavage from the pre-mRNA. At present it is not known how U5 loop 1 is positioned on the 5' exon prior to the first step of splicing. To address this question, we have used site-specific photoactivated crosslinking in yeast spliceosomes to investigate the interaction of U5 loop 1 with the pre-mRNA prior to the first step of splicing. We have found that the highly conserved uridines in loop 1 make ATP-dependent contacts with an approximately 8-nt region at the 5' splice site that includes the invariant GU. These interactions are dependent on functional U2 and U6 snRNAs. Our results support a model where U5 snRNA loop 1 interacts with the 5' exon in two steps during its targeting to the 5' splice site.

Adenosine Triphosphate↗
PubMed1999

Inosine and N1-methylinosine within a synthetic oligomer mimicking the anticodon loop of human tRNA(Ala) are major epitopes for anti-PL-12 myositis autoantibodies.

Sera of some patients afflicted with the inflammatory disease myositis contain antibodies of the anti-PL-12 type. A fraction of these polyclonal autoantibodies specifically precipitates the fully matured human tRNA(Ala) bearing the anticodon IGC (PL-12 antigen). Earlier work (Bunn & Mathews, 1987, Science 238:116-119) had shown that the epitopes are located entirely within the anticodon stem-loop of the tRNA(Ala). Here we demonstrate that human anti-tRNA(Ala) autoantibodies immunoprecipitate a synthetic polyribonucleotide containing inosine (I) and N1-methylinosine (m1I) separated by 2 nt as in the anticodon stem-loop of human tRNA(Ala). The shortest polyribonucleotide that can be immunoprecipitated corresponds to the pentanucleotide IpGpCpm1IpUp, which corresponds to part of the anticodon loop of human tRNA(Ala) and lacks the stem-loop structure. The efficiency of immunoprecipitation was about four times greater with longer polyribonucleotides capable of forming a stem-loop structure, and was abolished by altering the relative positions of I and m1I within the synthetic polynucleotide. Synthetic oligodeoxyribonucleotide analogs of the tRNA(Ala) stem-loop, containing the sequence dIpdGdCdm1Ip, are not antigenic. Our results show that human anti-tRNA(Ala) autoantibodies selectively recognize chemical details of modified nucleotides (the 6-keto group of inosine-34 and the 6-keto group and the N1-methyl groups of N1-methylinosine-37) within an anticodon loop structure of a tRNA molecule. We also describe the chemical synthesis of the phosphoramidite derivatives corresponding to N1-methylinosine and N1-methyl-2'-deoxyinosine for use in the automatic chemical synthesis of oligonucleotides containing N1-methylinosine and N1-methyl-2'-deoxyinosine.

Antibodies, Antiphospholipid↗