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PubMed2002

Exploring the potential of the monobody scaffold: effects of loop elongation on the stability of a fibronectin type III domain.

The tenth fibronectin type III domain of human fibronectin (FNfn10) is a small, monomeric beta-sandwich protein, similar to the immunoglobulins. We have developed small antibody mimics, 'monobodies', using FNfn10 as a scaffold. We initially altered two loops of FNfn10 that are structurally equivalent to two of the hypervariable loops of the immunoglobulin domain. In order to assess the possibility of utilizing other loops in FNfn10 for target binding, we determined the effects of the elongation of each loop on the conformational stability of FNfn10. We found that all six loops of FNfn10 allowed the introduction of four glycine residues while retaining the global fold. Insertions in the AB and FG loops exhibited very small degrees of destabilization, comparable to or less than predicted entropic penalties due to the elongation, suggesting the absence of stabilizing interactions in these loops in wild-type FNfn10. Insertions in the BC, CD and DE loops, respectively, resulted in modest destabilization. In contrast, the EF loop elongation was highly destabilizing, consistent with previous studies showing the presence of stabilizing interactions in this loop. These results suggest that all loops, except for the EF loop, can be used for engineering a binding site, thus demonstrating excellent properties of the monobody scaffold.

Amino Acid Sequence↗

PubMed1998

Lack of correlation between V3-loop peptide enzyme immunoassay serologic subtyping and genetic sequencing.

OBJECTIVE: To compare the performance of V3-loop peptide enzyme immunoassay (PEIA) methodologies from four different laboratories for subtyping HIV-1, and to determine the causes for the lack of correlation between V3-loop PEIA serotyping and subtyping by sequencing. MATERIALS AND METHODS: Synthetic peptides derived from the amino-acid consensus sequences of the V3-loop of group M strains representing genetic subtypes A-F as well as reference strains were evaluated in PEIA by four different laboratories for their ability to accurately determine the subtype in a panel of 85 sera obtained from persons infected with known HIV-1 subtypes (28 subtype A, 34 subtype B, four subtype C, 10 subtype D, seven subtype F, one each of subtype H and G). Furthermore, the V3 loop of the corresponding virus was compared with the V3 loop of the peptides used in PEIA. RESULTS: The correlation between HIV-1 subtyping by sequencing and V3-loop PEIA from the different laboratories varied considerably for the different HIV-1 subtypes: subtype A (46-68%), B (38-85%), C (75-100%), D (29-50%), and F (17-57%). A 70% agreement between PEIA and sequencing subtypes was observed for samples with the concordant presence of the same octameric sequences in the V3 loop of the virus and the V3 loop of the peptide used in PEIA; however, only 42% of specimens with different V3-loop octameric viral and peptide sequences yielded concordant results in V3-loop serotyping and genetic subtyping. CONCLUSION: Our results indicate that V3-loop PEIA methodologies used in different laboratories correlate poorly with genetic subtyping, and that their accuracy to predict HIV-1 subtypes in sera of Belgian individuals infected with different HIV-1 subtypes (A, B, C, D, F, G and H) vary considerably. The poor correlation between serotyping and genetic subtyping was partly due to the simultaneous occurrence of subtype-specific octameric sequences at the tip of the V3 loop of viruses belonging to different genetic subtypes.

Amino Acid Sequence↗

PubMed2005

Formation of loops in DNA under tension.

We study the formation of loops along a DNA molecule under applied tension, as might occur in single-DNA micromanipulation experiments with proteins which are able to simultaneously bind two DNA sites. We consider the case of "bare" DNA in the loop, which forms a "teardrop" shape, and the case where a single DNA-bending protein produces a "kink" in the middle of the loop; the presence of a right-angle kink in the loop reduces its bending energy by a factor of 3. Using the bending energy plus an estimate of the free energies associated with fluctuations and the elasticity of the extended nonlooped DNA, we obtain a probability distribution for loops as a function of loop size and force. Force strongly suppresses formation of all loops, but suppresses large loops more severely than small ones. This quenching effect of force is reduced in the presence of a kink in the loop. We also calculate the speed at which length is absorbed into loops between arbitrary positions along the DNA (i.e., for non-sequence-specific loop forming proteins). The speed of retraction of the molecule decays as a stretched exponential function of the force with characteristic force scales depending on the geometry of the loops.

Computer Simulation↗

PubMed1997

Is loop excision adequate primary management of adenocarcinoma in situ of the cervix?

OBJECTIVE: To assess the efficacy of cervical loop excision as primary management of adenocarcinoma in situ. DESIGN: A two-centre retrospective study. SETTING: Birmingham and Midlands Hospital for Women and City Hospital NHS Trust. POPULATION: Nineteen women with a histological diagnosis of adenocarcinoma in situ (high grade CIGN) of the cervix made on diathermy loop excision. MAIN OUTCOME MEASURES: Presence or absence of adenocarcinoma in situ at loop specimen margins, results of cervical cytological examinations following loop excision, results of histopathological assessment of any surgical specimens taken after initial loop excision. RESULTS: Of the 19 women with confirmed adenocarcinoma in situ on loop excision, the median age was 31, and 8 (42%) were nulliparous. The median follow up of these women was 19 months. Eleven women were treated by loop excision alone and have had normal follow up to 18 months. Five women have undergone further surgical procedures, four had a hysterectomy and one had a repeat loop excision. No residual disease was found in any of these surgical specimens, confirming that loop excision was adequate primary management of the disease. Three women have had abnormal endocervical follow up cytology suggestive of residual disease. One of these three women may represent a case of residual endocervical disease. Excision margins of the loop specimen were not involved by adenocarcinoma in situ in 11 women. However, excision margin status of the loop specimen did not appear to be predictive of residual disease. CONCLUSIONS: Our small retrospective study suggests that diathermy loop excision of the cervix is adequate primary management of adenocarcinoma in situ of the cervix. Cytological and colposcopic follow up, including cytobrush endocervical cytological sampling and long term follow up, is recommended in these women.

Adenocarcinoma↗

PubMed1997

Allele-specific adaptation of poliovirus VP1 B-C loop variants to mutant cell receptors.

Previous work has shown that three different mutations in domain 1 of the poliovirus receptor (Pvr), two in the predicted C'-C" ridge and one in the D-E loop, abolish binding of the P1/Mahoney strain. All three receptor defects could be suppressed by a mutation in the VP1 B-C loop of the viral capsid that was present in all 16 P1/Mahoney isolates adapted to the mutant receptors. To identify allele-specific mutations that enable poliovirus to utilize mutant receptors, and to understand the role of the VP1 B-C loop in adaptation, we selected mutant receptor-adapted viruses derived from two P1/Mahoney variants, one which lacks the VP1 B-C loop and one in which the VP1 B-C loop is replaced with the corresponding sequence from the P2/Lansing strain. Six adapted viral isolates were obtained after passage on mutant receptor-expressing cell lines. Sequence analysis revealed that each virus contained three to five mutations, and a total of 18 amino acid changes at 17 capsid residues were identified. Site-directed mutagenesis was used to evaluate the role of these mutations in adaptation to mutant Pvr. The results demonstrate that mutations in the viral canyon floor and rim are allele specific and compensate only for receptor defects in the C'-C" ridge of Pvr, suggesting that these sites interact in the virus-receptor complex. Furthermore, mutations in the VP1 E-F loop suppressed Pvr D-E loop defects, implying that the Pvr D-E loop contacts the VP1 E-F loop. Most of the other mutations mapped to interior capsid residues, some interacting with the fivefold- or threefold-related protomers. These mutations may regulate receptor interaction by controlling the structural flexibility of the viral capsid. In viruses lacking the VP1 B-C loop, single mutations were not sufficient to confer the adapted phenotype, in contrast to the 414 virus, which contains the B-C loop. Although the VP1 B-C loop appeared to be dispensable for adaptation, it may have provided a selective advantage in adaptation of P1/Mahoney to mutant Pvr.

Adaptation, Physiological↗

PubMed2005

Thick loop prostatectomy in the endoscopic treatment of benign prostatic hyperplasia: results of a prospective randomised study.

OBJECTIVE: The thick loop is a new device employed for transurethral resection of the prostate (TURP) using the standard resectoscope. The loop is broader and thicker than the standard one, resulting in better hemostasis because of its ability to cut, coagulate and vaporize tissue simultaneously. We evaluated the safety and efficacy of the thick loop device compared with the standard loop. PATIENTS AND METHODS: 103 patients with symptomatic benign prostatic hyperplasia were randomized to undergo either thick loop (51 patients) or standard loop TURP (52 patients). Patients were considered for surgery with the American Urological Association (AUA) symptom score greater than 7 and a maximum urinary flow rate <15 ml/s. Serum hemoglobin, hematocrit, electrolytes, operative time, prostate resected weight, catheterization time and complications were recorded. Twelve months later, the AUA score, maximum urinary flow rate (Q(max)) and postvoiding residual urine volume were evaluated. RESULTS: There were no significant differences between thick and standard loop TURP regarding the operative and catheterization time, prostate resection weight and postoperative levels of hemoglobin, hematocrit and electrolytes. Twelve months after TURP, the AUA score was significantly lower and Q(max) greater in the thick loop compared to the standard loop groups. CONCLUSIONS: Thick loop TURP is as safe as standard loop TURP with respect to blood loss, operative time and complications. Operating in a virtually bloodless field could allow a more radical TURP and provide an explanation for the better functional results (Q(max) and AUA score) obtained by the thick loop resection.

Aged↗

PubMed1969

Micropuncture study of water, electrolytes, and urea movements along the loops of henle in psammomys.

The mechanism by which the osmotic pressure increases in tubular fluid along the descending limb of the loop of Henle was examined in Psammomys undergoing salt diuresis. In two series of experiments, micropuncture samples were collected either from proximal and distal convolutions at the surface of the cortex, or from loops of Henle and collecting ducts at the surface of the extrarenal part of the papilla. Inulin-(3)H, urea-(14)C, Na(+), and K(+) concentrations, as well as osmotic pressure, were determined in all micropuncture samples.Net movements of water along the descending and ascending limbs of the loop could not be deduced by comparing inulin data obtained from convoluted tubules and from loops of Henle, since there appeared to be a large difference in the filtration rate of the superficial glomeruli (9 nl/min) and the deep ones (21.4 nl/min) under the conditions of these experiments. The results indicate that no large net movement of water occurred along the loop since a) only 23% of the filtrate was reabsorbed along the loop of Henle (including pars recta) of superficial nephrons despite the fact that all these loops reached markedly hypertonic regions; b) there was no positive correlation between (F/P)(In) in early distal samples and the simultaneous osmotic pressure of the urine; c) when (F/P)(In) and (F/P)(Osm) in loop samples were correlated, the increase in inulin concentration accounted only for 15% of the increase in osmotic pressure. Therefore, 85% of the concentrating process taking place along the descending limb must have resulted from net addition of solutes; this was directly supported by Na(+) and K(+) measurements in the loop samples, which showed that, at the tip of the loops, the Na(+) and K(+) flow rates were correlated to the osmotic pressure. Moreover, since the load of Na(+) urea flow rate in superficial early distal tubules was constant and independent of the urinary osmotic pressure, it is suggested that a medullary recycling of both ions occurred between ascending and descending limbs.Urea-(14)C concentration in the loop samples indicates a net addition of urea into the descending limb; the mean and K(+) delivered to the distal superficial tubules was 4.18 times its filtration rate, suggesting a recycling of urea from collecting ducts to Henle's loops. The permeability properties of the wall of the thin descending limb are discussed in relation to the obtained results.

Animals↗

PubMed2004

Use of a structural alphabet for analysis of short loops connecting repetitive structures.

BACKGROUND: Because loops connect regular secondary structures, analysis of the former depends directly on the definition of the latter. The numerous assignment methods, however, can offer different definitions. In a previous study, we defined a structural alphabet composed of 16 average protein fragments, which we called Protein Blocks (PBs). They allow an accurate description of every region of 3D protein backbones and have been used in local structure prediction. In the present study, we use this structural alphabet to analyze and predict the loops connecting two repetitive structures. RESULTS: We first analyzed the secondary structure assignments. Use of five different assignment methods (DSSP, DEFINE, PCURVE, STRIDE and PSEA) showed the absence of consensus: 20% of the residues were assigned to different states. The discrepancies were particularly important at the extremities of the repetitive structures. We used PBs to describe and predict the short loops because they can help analyze and in part explain these discrepancies. An analysis of the PB distribution in these regions showed some specificities in the sequence-structure relationship. Of the amino acid over- or under-representations observed in the short loop databank, 20% did not appear in the entire databank. Finally, predicting 3D structure in terms of PBs with a Bayesian approach yielded an accuracy rate of 36.0% for all loops and 41.2% for the short loops. Specific learning in the short loops increased the latter by 1%. CONCLUSION: This work highlights the difficulties of assigning repetitive structures and the advantages of using more precise descriptions, that is, PBs. We observed some new amino acid distributions in the short loops and used this information to enhance local prediction. Instead of describing entire loops, our approach predicts each position in the loops locally. It can thus be used to propose many different structures for the loops and to probe and sample their flexibility. It can be a useful tool in ab initio loop prediction.

Computational Biology↗

PubMedDate not supplied

[Closed loop intestinal obstruction: role of computerized tomography].

PURPOSE: The obstruction of a bowel segment at both ends results in a closed loop obstruction. Progression to strangulation frequently occurs if surgical intervention is delayed. The role of plain radiography in the diagnosis of closed loop obstruction and strangulation has been shown to be limited, while the recent literature has demonstrated the growing role of computed tomography (CT). This paper reports our experience in the study of closed loop obstruction by CT. MATERIAL AND METHODS: The CT studies of 12 patients with surgically confirmed closed loop obstruction were retrospectively reviewed. The following CT signs were used for the diagnosis: a) fluid-filled distended loops, b) C-shaped incarcerated loop, c) radial distribution of several dilated bowel loops and mesenteric vessels converging toward the point of obstruction, d) triangular or fusiform tapering of the closed loop and/or whirl sign in the site of obstruction. RESULTS: On the basis of these signs, the diagnosis was made in 11 of 12 patients. Only 1 patient, who had a negative CT study, was positive at a subsequent enteroclysis. CT findings of strangulation were associated in 3 cases: slight wall thickening with vascular congestion and mesenteric ascites, confirmed at surgery. DISCUSSION AND CONCLUSIONS: Small bowel obstruction can be distinguished into simple and closed loop obstructions. The latter is a more severe condition which is often complicated by strangulation with vascular impairment, edema and intramural and mesenteric hemorrhage. Consequent arterial insufficiency rapidly leads to ischemia, infarction and necrosis. The radiologist plays a role in the early recognition of the closed loop obstruction and of any sign of strangulation. The role of CT in the diagnosis and workup of patients with suspected intestinal occlusion has been analyzed in the literature with reported 63% sensitivity, 78% specificity and 66% accuracy. CT is also capable of revealing the causes of occlusion in 73-95% of cases. The above CT signs, as confirmed in our experience, allow to identify closed loop obstruction and also small bowel strangulation, thus supplying a valuable contribution to diagnosis and accurate preoperative evaluation. We conclude that CT can accurately demonstrate the presence of closed loop obstruction and can be the technique of choice in patients in whom obstruction is associated with clinical signs suggestive of strangulation.

Female↗

PubMed1987

Bacterial overgrowth and mucosal changes in isolated (Thirty-Vella) ileal loops in rabbits. Effects of intraluminal antibiotics.

Atrophy of villi, with increases in crypt depth and Paneth cell number and size, occurs in chronically isolated (Thirty-Vella) ileal loops in rabbits. These loops are known to be heavily colonized with aerobic bacteria. To study the possible effect of the bacterial overgrowth, 2 experiments were performed. In the first study, two isolated ileal loops were created in each of 14 rabbits. The antibiotic loop was flushed with nonabsorbable antibiotics (neomycin, bacitracin, and gentamycin), whereas the control loop was flushed with saline. The antibiotic solution achieved a reduction in bacterial growth as compared to the loops flushed with saline. In the second study, a single ileal loop was created in each of 20 rabbits. Loops of 11 animals were flushed with an absorbable antibiotic (cefoxitin) and gentamycin, whereas those in 9 other rabbits were flushed with saline. This antibiotic combination achieved an essentially sterile loop. In both experiments, the Paneth cell population and crypt depth were less in antibiotic loops as compared to saline loops, whereas the degree of villus atrophy was nearly equal. These studies suggest a link between the overgrowth of bacteria seen in these isolated loops and the morphologic changes in the crypts.

Animals↗

PubMed1981

Glycoprotein degradation in the blind loop syndrome: identification of glycosidases in jejunal contents.

Contents obtained from jejunum of normal controls, self-emptying and self-filling blind loop rats were analyzed for the presence of glycoprotein-degrading glycosidases. The blind loop syndrome was documented by the increased fat excretion and slower growth rate of self-filling blind loop rats 6 wk after surgery. With p-nitrophenylglycosides as substrate, the specific activity of alpha-N-acetylgalactosaminidase, a potential blood group A destroying glycosidase, was 0.90+/-0.40 mU/mg of protein. This level was 23-fold higher than the specific activity of normal controls. In partially purified self-filling blind loop contents, the activity of alpha-N-acetylgalactosaminidase was 9- to 70-fold higher than activities of self-emptying and normal controls. Antibiotic treatment with chloromycetin and polymyxin decreased 24-fold the glycosidase levels in self-filling blind loops. In experiments with natural substrate, the blood group A titer of a20,000g supernate from normal jejunal homogenates decreased 128-fold after 24-h incubation with blind loop contents. Normal contents failed to diminish the blood group reactivity of the natural substrate. Furthermore, blind loop contents markedly decreased the blood group A titer of isolated brush borders. Incubation between blind loop bacteria and mucosal homogenates or isolated brush borders labeled with d-[U-(14)C]glucosamine revealed increased production of labeled ether extractable organic acids. Likewise, intraperitoneal injection of d-[U-(14)C]glucosamine into self-filling blind loop rats resulted in incorporation of the label into luminal short chain fatty acids. These results suggest that glycosidases may provide a mechanism by which blind loop bacteria obtain sugars from intestinal glycoproteins. The released sugars are used and converted by bacteria into energy and organic acids. This use of the host's glycoproteins would allow blind loop bacteria to grow and survive within the lumen independent of exogenous sources.

Animals↗

PubMed1995

The energy of formation of internal loops in triple-helical collagen polypeptides.

The sequence-dependent local destabilization in the interior of the collagen triple helix has been evaluated by means of conformational energy computations. Using a model poly(Gly-Pro-Pro) triple helix as the reference state, a method was developed for generating local loops, i.e., internal deformations, and analyzing their conformations. A seven-residue Gly-Pro-Pro- Gly-Pro-Pro-Gly fragment was replaced by the Gly-Pro-Ala-Gly-Ala-Ala-Gly sequence in one, two, or all three of the strands of the loop region. A set of loop conformations was generated in which the ends of the loop were initially fixed in the triple-helical structure. The potential energy of the entire deformed triple helix was then minimized, resulting in a variety of structures that contained deformed loops. The conformations of the triple helices at the two ends of the loops remained essentially unchanged in many of the low-energy conformations. In numerous high-energy conformations, however, the triple-helical segments were also partially or totally disrupted. The minimum-energy conformations of the whole structures were compared in terms of rms deviations of atomic coordinates with respect to the original triple helix, and of the shapes of the loops (using a distance function derived from differential geometry). Three new geometrical parameters-stretch S, kink K, and unwinding U-were defined to describe the changes in the overall orientation of the triple helices at the two ends of the loop. It is shown that, when the number of Pro residues in a short fragment is reduced, the triple helical structure can accomodate internal loops (i.e., distortions) within a 5 kcal/mol cutoff from the essentially unperturbed triple helical structure. For structures with a Gly-Pro-Ala-Gly-Ala-Ala-Gly sequence in all three strands, the probability of finding conformations with internal loops is small, i.e., 0.06. Internal loops affect the overall orientation of these structures, as measured by the helix-distortion parameters S, K, and U.

Amino Acid Sequence↗

PubMed1990

The size of chromatin loops in HeLa cells.

It is widely believed that the chromatin fibre is organized into loops during interphase, with the loop being implicated as an important unit of nuclear function. However, there remains little direct evidence for looping, with estimates of loop size varying widely. This has led to the suggestion that some loops, or even all of them, arise artefactually during isolation as chromatin aggregates so easily. We have now investigated the effect of isolation procedure on loop size using HeLa cells encapsulated in agarose to allow easy manipulation. Loop size in various derivatives (i.e. nuclei, nucleoids, matrices and scaffolds) critically depended on procedure; some (or all) of their loops are artefacts. The loop size in derivatives isolated using the most 'physiological' conditions was 86 kb; this remained unchanged throughout the cell cycle. This loop size is probably an average of a range of loops of between 5 and 200 kb.

Cell Cycle↗

PubMed2001

Optimization of solvation models for predicting the structure of surface loops in proteins.

A novel procedure for optimizing the atomic solvation parameters (ASPs) sigma(i) developed recently for cyclic peptides is extended to surface loops in proteins. The loop is free to move, whereas the protein template is held fixed in its X-ray structure. The energy is E(tot) = E(FF)(epsilon = nr) + summation operator sigma(i)A(i), where E(FF)(epsilon = nr) is the force-field energy of the loop-loop and loop-template interactions, epsilon = nr is a distance-dependent dielectric constant, and n is an additional parameter to be optimized. A(i) is the solvent-accessible surface area of atom i. The optimal sigma(i) and n are those for which the loop structure with the global minimum of E(tot)(n, sigma(i)) becomes the experimental X-ray structure. Thus, the ASPs depend on the force field and are optimized in the protein environment, unlike commonly used ASPs such as those of Wesson and Eisenberg (Protein Sci 1992;1:227-235). The latter are based on the free energy of transfer of small molecules from the gas phase to water and have been traditionally combined with various force fields without further calibration. We found that for loops the all-atom AMBER force field performed better than OPLS and CHARMM22. Two sets of ASPs [based on AMBER (n = 2)], optimized independently for loops 64-71 and 89-97 of ribonuclease A, were similar and thus enabled the definition of a best-fit set. All these ASPs were negative (hydrophilic), including those for carbon. Very good (i.e., small) root-mean-square-deviation values from the X-ray loop structure were obtained with the three sets of ASPs, suggesting that the best-fit set would be transferable to loops in other proteins as well. The structure of loop 13-24 is relatively stretched and was insensitive to the effect of the ASPs.

Hydrogen↗

PubMed2006

Ab initio computational modeling of loops in G-protein-coupled receptors: lessons from the crystal structure of rhodopsin.

With the help of the crystal structure of rhodopsin an ab initio method has been developed to calculate the three-dimensional structure of the loops that connect the transmembrane helices (TMHs). The goal of this procedure is to calculate the loop structures in other G-protein coupled receptors (GPCRs) for which only model coordinates of the TMHs are available. To mimic this situation a construct of rhodopsin was used that only includes the experimental coordinates of the TMHs while the rest of the structure, including the terminal domains, has been removed. To calculate the structure of the loops a method was designed based on Monte Carlo (MC) simulations which use a temperature annealing protocol, and a scaled collective variables (SCV) technique with proper structural constraints. Because only part of the protein is used in the calculations the usual approach of modeling loops, which consists of finding a single, lowest energy conformation of the system, is abandoned because such a single structure may not be a representative member of the native ensemble. Instead, the method was designed to generate structural ensembles from which the single lowest free energy ensemble is identified as representative of the native folding of the loop. To find the native ensemble a successive series of SCV-MC simulations are carried out to allow the loops to undergo structural changes in a controlled manner. To increase the chances of finding the native funnel for the loop, some of the SCV-MC simulations are carried out at elevated temperatures. The native ensemble can be identified by an MC search starting from any conformation already in the native funnel. The hypothesis is that native structures are trapped in the conformational space because of the high-energy barriers that surround the native funnel. The existence of such ensembles is demonstrated by generating multiple copies of the loops from their crystal structures in rhodopsin and carrying out an extended SCV-MC search. For the extracellular loops e1 and e3, and the intracellular loop i1 that were used in this work, the procedure resulted in dense clusters of structures with Calpha-RMSD approximately 0.5 angstroms. To test the predictive power of the method the crystal structure of each loop was replaced by its extended conformations. For e1 and i1 the procedure identifies native clusters with Calpha-RMSD approximately 0.5 angstroms and good structural overlap of the side chains; for e3, two clusters were found with Calpha-RMSD approximately 1.1 angstroms each, but with poor overlap of the side chains. Further searching led to a single cluster with lower Calpha-RMSD but higher energy than the two previous clusters. This discrepancy was found to be due to the missing elements in the constructs available from experiment for use in the calculations. Because this problem will likely appear whenever parts of the structural information are missing, possible solutions are discussed.

Computer Simulation↗

PubMed1996

A comparison of end and loop colostomy for fecal diversion in gynecologic patients with colonic fistulas.

OBJECTIVE: The purpose of this study was to evaluate the early morbidity, stomal complications, and overall survival rate of gynecologic oncology patients treated with end or loop colostomy for management of colonic fistula. METHODS: Seventy-five women with fistulas undergoing fecal diversion procedures from 1983 to 1993 were identified. Information relevant to tumor history, type of fistula, operative procedure, complications, and outcome was extracted from the medical records. RESULTS: A recto- or sigmoid-vaginal fistula was present in 55 patients. The remaining 20 patients had either a colonic fistula involving the bladder, uterus, or skin or multiple fistulas. In 66 (88%) patients, the pelvis had been irradiated. Loop colostomy was performed in 47 (63%) patients and end colostomy in 28 (37%) of patients. Cancer was present in 31 (66%) of 47 patients undergoing loop colostomy compared with 12 (43%) of 28 patients undergoing end colostomy (P = 0.05). No significant differences were detected with regard to age at cancer diagnosis or age at the time of colostomy, body habitus, medical condition, type of cancer, history of pelvic irradiation, or incidence of multiple or complex fistulas. Median operative time was increased by 1 hr and median blood loss from 50 to 200 ml with end colostomy. No significant advantage of loop colostomy over end colostomy was detected in the incidence of acute morbidity or hospital stay. Early stomal complications occurred in 8 patients, 7 of whom had loop colostomy (P = 0.25). Delayed complications (prolapse, retraction, stool per vagina, or fistula) following loop colostomies occurred more frequently than following end colostomy: 12 (26%) of 47 compared with 2 (7%) of 28 (P = 0.05), respectively. Five loop stomas were later converted to end stomas. In addition only one of four attempted loop colostomy closures was successful. Overall survival in both groups of patients was similar. CONCLUSION: There were no statistically significant differences between patients treated with loop or end colostomy with regard to early morbidity or survival. The early and frequent occurrences of stomal complications after loop colostomy overshadow the clinically minor advantages of this method of fecal diversion. Although loop colostomy was performed more often in this setting, these data do not support its routine use for the management of gynecologic oncology patients with fistulas.

Colonic Diseases↗

PubMed1998

GlnK, a PII-homologue: structure reveals ATP binding site and indicates how the T-loops may be involved in molecular recognition.

GlnK is a recently discovered homologue of the PII signal protein, an indicator of the nitrogen status of bacteria. PII occupies a central position in the dual cascade that regulates the activity of glutamine synthetase and the transcription of its gene. The complete role of Escherichia coli GlnK is yet to be determined, but already it is known that GlnK behaves like PII and can substitute for PII under some circumstances thereby adding to the subtleties of nitrogen regulation. There are also indications that the roles of the two proteins differ; the expression of PII is constitutive while that of GlnK is linked to the level of nitrogen in the cell. The discovery of GlnK begs the question of why E. coli has both GlnK and PII. Clearly, the structural similarities and differences of GlnK and PII will lead to a better understanding of how PII-like proteins function in E. coli and other organisms. We have crystallised and solved the X-ray structure of GlnK at 2.0 A resolution. The asymmetric unit has two independent copies of the GlnK subunit and both pack around 3-fold axes to form trimers. The trimers have a barrel-like core with recognition loops (the T-loops) that protrude from the top of the molecule. The two GlnK molecules have similar core structures to PII but differ significantly at the C terminus and the loops. The T-loops of the two GlnK molecules also differ from each other; one is disordered while the conformation of the other is stabilised by lattice contacts. The conformation of the ordered T-loop of GlnK differs from that observed in the PII structure despite the fact that their sequences are very similar. The structures suggest that the T-loops do not have a rigid structure and that they may be flexible in solution. The presence of a turn of 310 helix in the middle of the T-loop suggests that secondary structure could form when it interacts with soluble receptor enzymes.Co-crystals of GlnK and ATP were used to determine the structure of the complex. In these crystals, GlnK occupies a position of 3-fold symmetry. ATP binds in a cleft on the side of the molecule. The cleft is suitably positioned for ATP to influence the flexible T-loops. It is found at the junction of two beta sheets and is formed by two peptides one of which contains a variant of the "Gly-loop" found in other mononucleotide binding proteins. This sequence, Thr-Gly-X-X-Gly-Asp-Gly-Lys-Ile-Phe, forms part of the B-loop and is conserved in a wide variety of organisms that include bacteria, algae and archeabacteria. This sequence is more highly conserved than the functional T-loop, suggesting that ATP has an important role in PII-like proteins.

Adenosine Triphosphate↗

PubMed1991

Complexes formed by complementary RNA stem-loops. Their formations, structures and interaction with ColE1 Rom protein.

Regulation of replication of plasmid ColE1 involves interaction of two plasmid-specified RNA transcripts. One of these RNAs (RNA II) serves as a primer for DNA synthesis, and the other (RNA I) is complementary to part of RNA II. The complementary regions of RNA I and RNA II form several stem-loop structures. Binding of these RNAs that regulates DNA replication begins by interaction at the loop regions. Plasmid-coded Rom protein stabilizes the product of the interaction. In this paper, the mechanism of the loop-to-loop interaction between pairs of RNA stem-loops having various nucleotide sequences is studied. Binding of two stem-loops containing six to eight nucleotides in their loops requires that the loop sequences be complementary, whereas the stem sequences need not be. The association rate constants for binding of complementary pairs with various sequences are relatively similar, around 1 x 10(6) M-1 S-1. On the other hand, the rates of dissociation of the complexes vary greatly depending on the loop sequence, even for complexes having the same base composition, suggesting a strong effect of base-stacking. All the complementary bases in the seven-nucleotide loops participate in complex formation, and the resulting complex is bent a little at the interacting region. Rom binds and stabilizes any complex formed by pairs containing fully complementary loop sequences. Structures are proposed for the RNA complexes with and without Rom.

Bacterial Proteins↗