Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Ligation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 235 records · Page 13Linked to original sources
PubMed2006

Intraoral duct ligation without inclusion of the parasympathetic nerve supply induces rat submandibular gland atrophy.

The atrophic effect of ligating the main duct of the right submandibular gland was examined in rat using a novel intraoral approach that did not include the chorda lingual (CL) nerve. Comparison was made with the effect of duct ligation including the attached CL nerve as carried out in previous studies. In all animals, the contralateral, unligated left submandibular gland was used as a control. At different times (1, 2, 7, 14 and 21 days) after ligation, glands were removed and weighed. Tissue was fixed for morphological analysis and homogenized for biochemical assay of secretory proteins. After 21 days, ligated glands showed a significant decrease in wet weight compared with unligated glands. Weight loss was the greatest (P < 0.05) in glands ligated with the CL nerve included. Light microscopy revealed that following ligation, an initial inflammatory reaction was followed by severe atrophy of acini and granular ducts. The atrophy was less severe when the CL nerve was not ligated. Secretory proteins were decreased from day 1 onwards following duct ligation in both groups. It can be concluded that most of the atrophy induced by duct ligation is independent of damage caused to the parasympathetic nerve supply, although the latter causes a greater atrophy presumably due to denervation.

Animals↗
PubMed2005

Meta-analysis: endoscopic variceal ligation for primary prophylaxis of oesophageal variceal bleeding.

BACKGROUND: The treatment effects of primary prophylactic endoscopic variceal ligation are unclear. AIM: To compare the treatment effects of endoscopic variceal ligation and beta-blockers for primary prophylaxis of oesophageal variceal bleeding. In addition, a subgroup analysis was done with the purpose to delineate differences in the effects of intervention that were biologically based. METHODS: We performed a literature search for randomized controlled trials, which compared the treatment effects of endoscopic variceal ligation with beta-blockers for primary prophylaxis of oesophageal variceal bleeding. Of the 955 articles screened, eight randomized-controlled trials including 596 subjects (285 with endoscopic variceal ligation and 311 with beta-blockers) were analysed. Outcomes measures evaluated were first gastrointestinal bleed, first variceal bleed, all-cause deaths, bleed-related deaths and severe adverse events. The measure of association employed was relative risk; with heterogeneity and sensitivity analyses. RESULTS: Variceal obliteration was obtained in 261 (91.6%) patients and target beta-blockers therapy was achieved in 294 (94.5%) patients (P = 0.19). Endoscopic variceal ligation compared with beta-blockers significantly reduced rates of first gastrointestinal bleed by 31% (RR, 0.69; 95% CI: 0.49-0.96; P = 0.03; NNTB = 15) and first variceal bleed by 43% (RR, 0.57; 95% CI: 0.38-0.85; P = 0.0067; NNTB = 11). All-cause deaths and bleed-related deaths were unaffected (RR, 1.03; 95% CI: 0.79-1.36; P = 0.81 and RR, 0.84; 95% CI: 0.44-1.61; P = 0.60 respectively). Severe adverse events were significantly less in endoscopic variceal ligation compared with beta-blockers (RR, 0.34; 95% CI: 0.17-0.69; P = 0.0024; NNTB = 28). Sensitivity analysis of five trials published in peer review journals and four trials with high quality showed results similar to those seen in the primary analysis of all the eight trials, confirming stability of conclusions. Following variceal obliteration with endoscopic variceal ligation, oesophageal varices recurred in 83 (29.1%) patients. Seven (28.1%) patients bled with one fatal outcome. In subgroup analyses, endoscopic variceal ligation had significant advantage compared wtih beta-blockers in trials including < or =30% patients with alcoholic cirrhosis, >30% patients with Child Class C cirrhosis and >50% patients with large varices. CONCLUSIONS: In patients with cirrhosis with moderate to large varices and who have not bled, endoscopic varices ligation compared with beta-blockers significantly reduced bleeding episodes and severe adverse events, but had no effect on mortality.

Adrenergic beta-Antagonists↗
PubMed2006

Systemic mediators induce fibrogenic effects in normal liver after partial bile duct ligation.

BACKGROUND/AIMS: Collagen production by activated hepatic stellate cells (HSCs) is a key event in liver fibrosis, and a number of factors have been characterized that trigger HSC activation and collagen production. However, it remains unclear if these factors act locally at the site of injury or also affect HSCs distant to the site of injury. METHODS: A model of partial bile duct ligation (PBDL) in which fibrogenesis can be compared between the injured ligated lobe and the non-ligated lobe. RESULTS: After PBDL, HSCs showed an increased expression of procollagen type I alpha1 mRNA and collagen-reporter gene activity not only in the ligated lobe, but also in the non-ligated lobe, albeit at a lower level. In contrast, an increase in the number of desmin- and alpha-smooth muscle actin positive HSCs, and accumulation of inflammatory cells were observed only in the ligated lobe. Although transforming growth factor-beta (TGF-beta) mRNA was increased only in the ligated lobe, Smad2/3 were activated in the ligated and the non-ligated lobe. These data suggest that the systemic increase in profibrogenic mediators including TGF-beta induces collagen transcription in the uninjured liver. CONCLUSION: Systemic profibrogenic mediators from the injury site affect the residual non-injured liver.

Actins↗
PubMedDate not supplied

Transdiaphragmatic approach to thoracic duct ligation in the cat.

An approach combining ventral midline celiotomy with transdiaphragmatic thoracotomy was evaluated in eight healthy cats for ligation of the thoracic duct system. Evans Blue solution was injected into the right colic lymph node to outline the intestinal lymphatic trunk and the thoracic duct system. Three cats (group 1) had mesenteric lymphangiograms and three (group 2) had only lymph node dye injection before thoracic duct ligation. The thoracic duct system was ligated with hemostatic clips just cranial to the aortic hiatus of the diaphragm, through a left transdiaphragmatic thoracotomy. Two cats (group 3) had prethoracotomy mesenteric lymphangiograms and thoracic duct isolation without ligation. Mesenteric lymphangiography was performed immediately after the surgery. In all of the cats, an absence of contrast medium in the thoracic duct system cranial to the surgical site was interpreted as complete obstruction. Four weeks after ligation, there was complete obstruction of the thoracic duct system with alternate lymphaticovenous communications in four of the six cats with ligated thoracic duct systems. Partial obstruction of the thoracic duct system with alternate lymphaticovenous communications was present in the other two cats. Both cats without thoracic duct ligation had patent thoracic duct systems. At necropsy of the six cats with ligated thoracic ducts, there was mild focal lymphadenitis of injected lymph nodes in three cats. The wall of the aorta adjacent to the hemostatic clips was normal in all six cats. The surgical technique was simple and provided excellent exposure. Vital staining with Evans Blue helped visualize the thoracic duct system, but mesenteric lymphangiography did not. Postligation lymphangiography was not of value in identifying incomplete ligation.

Animals↗
PubMedDate not supplied

Long-term results of complete and partial ligation of congenital portosystemic shunts in dogs.

The medical records of 65 dogs that underwent complete or partial ligation of a single congenital portosystemic shunt (CPSS) were reviewed to determine the long-term clinical clinical results. Information retrieved from the records included age at surgery, preligation (baseline) portal pressure, postligation portal pressure, change in portal pressure from baseline, complete or partial occlusion of the shunting vessel and fasting, and 2-hour postprandial bile acids from the preoperative, early postoperative (PO), and greater than 1 year PO time periods. A clinical rating score derived from a follow-up examination greater than 1 year PO was assigned to each dog. Of the 56 dogs that survived the perioperative period, 29 (52%) had complete and 27 (48%) had partial ligations. Age at surgery, pre- and postligation portal pressure, change in portal pressure from baseline and serum bile acid concentrations were not related to long-term clinical outcome. Clinical rating scores were significantly greater for dogs with partial CPSS ligations compared with dogs with complete ligations, indicating a less favorable clinical outcome for partial ligations. Fasting and 2-hour postprandial bile acid values at both PO time intervals were significantly greater in partial versus complete ligation groups. Follow-up information for more than 1 year was available on 18 of 29 dogs (62%) with complete ligations. All were clinically normal. Of 27 dogs with partial ligations, 11 dogs (41%) developed recurrence of clinical signs resulting in presentation to the university or referring veterinarian for additional surgery, medical management, or euthanasia. Only three dogs with partial CPSS ligation (11%) were clinically normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1973

Effect of carotid ligation on cerebral blood flow in baboons. I. Response to altered arterial PCO2.

Measurements of cerebral blood flow (CBF) were made in anaesthetized baboons before and after ipsilateral carotid artery ligation and also after bilateral carotid ligation. Results showed that at normocapnia (PaCO(2) 38-39 mmHg) there was little change in cerebral blood flow on ipsilateral carotid ligation, but when both carotid arteries were tied the blood flow to the brain fell significantly. At hypercapnia (PaCO(2) 58-60 mmHg) the CBF/CO(2) gradient fell significantly on ipsilateral carotid ligation; on bilateral carotid ligation there was only minimal rise in cerebral blood flow in response to the rise in PaCO(2). At hypocapnia (PaCO(2) 20-21 mmHg) the gradients of fall in the CBF were similar before and after ipsilateral carotid ligation; after bilateral carotid ligation there was minimal change in the CBF in response to the fall in the PaCO(2). It is suggested that, although cerebral blood flow may be normal after ipsilateral carotid ligation, the circulatory reserve of the brain is not sufficient to meet physiological challenges. This may be the reason for the development of delayed neurological complications after carotid artery ligation.

Animals↗
PubMed1981

Acute changes of myocardial norepinephrine and glycogen phosphorylase in ischemic and non-ischemic areas after coronary ligation in dogs.

The myocardial norepinephrine (NE) content and glycogen phosphorylase activity were measured before and after coronary artery ligation in dogs anesthetized with pentobarbital. Coronary artery ligation was performed by ligating a branch of the left anterior descending coronary artery (LAD). Before coronary artery ligation, the NE content in the circumflex area (778.05 +/- 37.83 ng.g-1 wet tissue) was significantly higher than that in the LAD area (630.57 +/- 35.75 ng.g-1 wet tissue). The NE content in the circumflex area (non-ischemic area) decreased significantly 1.5, 3, 30. 60, and 420 min after coronary artery ligation, while that in the LAD area (ischemic area) did not decrease significantly except for the NE level obtained after 420 min of coronary artery ligation. The glycogen phosphorylase activity increased significantly 1.5 min after coronary artery ligation in both non-ischemic and ischemic areas. From the foregoing results, it is suggested that NE release occurs in both ischemic and non-ischemic areas within at least 1.5 min after coronary artery ligation, and that within 60 min after coronary artery ligation the NE content decreases in the non-ischemic area but not in the ischemic area probably because of very limited washout of the released NE in the ischemic area.

Animals↗
PubMed1981

Late complications of carotid ligation in the treatment of intracranial aneurysms.

The results in 57 consecutive patients treated with carotid ligation for an intracranial aneurysm were analyzed to define the risks of late complications. The average age for the group was 46 years. Eighty percent of the aneurysms were located on the internal carotid artery (ICA). The acute morbidity and mortality of the treatment and the natural history of the disease was 29%. Twenty-four patients were discharged with a common carotid artery (CCA) ligation, and 21 patients with an ICA ligation. Three patients from each ligation group could not be located for review. The follow-up period for the 21 patients with CCA ligation ranged from 1 to 15 years, with an average of 8.4 years, and for the 18 patients with ICA ligation it ranged from 2 to 19.5 years, with an average of 12.5 years. Excluding deaths from unrelated causes, five of the 21 patients with CCA ligation developed a late complication. Two patients had a transient ischemic attack (TIA). Two patients had a subarachnoid hemorrhage (SAH), one of which was fatal and was preceded by a TIA. Two patients developed monocular blindness. None of the patients had a stroke. Excluding deaths from unrelated causes, five of the 18 patients with ICA ligation developed a late complication. One patient had a fatal SAH. Three patients had a TIA, two of which were followed by a stroke. One patient had a stroke in the cerebral hemisphere contralateral to the side of the carotid ligation.

Adult↗
PubMed1995

Endoscopic ligation compared with sclerotherapy for treatment of esophageal variceal bleeding. A meta-analysis.

PURPOSE: To compare the effect of endoscopic ligation with that of sclerotherapy in the treatment of patients with bleeding esophageal varices. DATA SOURCES: Strategies to identify published and unpublished research included searches of computerized bibliographic and scientific citations, review of citations in relevant primary articles, searches of services providing information on unpublished studies, contact with primary investigators and the ligation equipment manufacturer, and review of proceedings from pertinent scientific meetings. STUDY SELECTION: From 158 potentially relevant articles, duplicate independent review identified 7 relevant randomized trials that compared endoscopic ligation with sclerotherapy for the treatment of patients with bleeding esophageal varices. DATA ABSTRACTION: Independent, duplicate data abstraction of the population, intervention, outcome, and methodologic quality of the trials was done. DATA SYNTHESIS: Ligation therapy compared with sclerotherapy reduced the rebleeding rate (odds ratio, 0.52 [95% CI, 0.37 to 0.74]), the mortality rate (odds ratio, 0.67 [CI, 0.46 to 0.98]), and the rate of death due to bleeding (odds ratio, 0.49 [CI, 0.24 to 0.996]). Four patients would need to be treated with ligation instead of sclerotherapy to avert one rebleeding episode, and 10 would need to be treated with ligation instead of sclerotherapy to prevent one death. Esophageal strictures occurred less frequently with ligation (odds ratio, 0.10 [CI, 0.03 to 0.29]), but no significant differences were seen between treatments for pulmonary infections or bacterial peritonitis. Additionally, the number of endoscopic treatment sessions required to achieve variceal obliteration was lower with ligation than with sclerotherapy. CONCLUSIONS: On the basis of lower rates of rebleeding, mortality, and complications and the need for fewer endoscopic treatments, ligation should be considered the endoscopic treatment of choice for patients with esophageal variceal bleeding.

Cause of Death↗
PubMed1999

[Elastic band ligation in secondary prevention of esophageal varices hemorrhage].

BACKGROUND: Sclerotization of esophageal varices may result in serious esophageal destruction. Elastic band ligation of esophageal varices is recently used in similar indications. AIM: The aim of the study was to evaluate in a pilot study the efficacy and safety of elastic band ligation for secondary prevention of bleeding from esophageal varices in patients who experienced life-threatening and were not yet treated with elastic band ligation. PATIENTS AND METHODS: Saeed's method of elastic band ligation was used in 10 patients (5 men and 5 women) aged from 16-60, who had already experienced life-threatening esophageal varices hemorrhage. One patient awaiting liver transplantation underwent preventive ligation. RESULTS: Esophageal varices were eradicated in 5 patients, minimum range residual varices remained in 4 patients after ligation. Haemorrhage recurrence was observed in 4 patients and was successfully treated by single urgent sclerotization. Two patients developed neo-varices. The ligation program was stopped in one female patient with mitral stenosis and tricuspidal insufficiency because of stenocardias occurring during ligation. In one session, 4.5 elastic bands in average were applied. The treatment of one patient required 4 sessions during 2.5 months in average. CONCLUSION: Elasic band ligation is an effective and safe method for the treatment of esophageal varices. Short term results are promising. (Tab. 1, Fig. 1, Ref. 18.)

Adolescent↗
PubMed1991

Main pulmonary artery ligation reduces lung fluid production in fetal sheep.

Pulmonary hypoplasia is increasing as a cause of neonatal death. To understand the pathophysiology of pulmonary hypoplasia, the physiology of fetal lung growth must first be understood. Lung fluid production and fetal breathing are primary factors regulating lung growth. Interruption of pulmonary arterial flow also decreases fetal lung growth. To define the relationship of pulmonary arterial flow to other factors known to be important for fetal lung growth, breathing and lung fluid production were measured after postductal main pulmonary artery (MPA) ligation in fetal sheep. Surgical preparation at 107-116 d gestation included placement of vascular catheters and a tracheal catheter connected to an intrauterine collection bag for lung fluid. Five fetuses served as monitored controls (catheters only), 3 as sham operated controls (catheters and thoracotomy), and 7 had MPA ligation. MPA ligation significantly decreased lung weights at 131-140 d; mean dry weight (g): MPA ligation--6.7, sham--23.4, monitored--22.3. Mean rates of lung fluid production (mL/h) were also decreased (d gestation): 116-122 d: MPA ligation--2.2, sham--9.1, monitored--6.8; 123-129 d: MPA ligation--2.1, sham--9.1, monitored--6.2; 130-136 d: MPA ligation--1.5, sham--12.4, monitored--7.7. There were no differences between MPA ligated, sham, and monitored fetuses in the incidence or intensity of fetal breathing movements. Decreased lung fluid production after main pulmonary artery ligation is most likely due to decreased secretion of lung fluid. Pulmonary arterial flow in other models of pulmonary hypoplasia which decrease lung fluid production (i.e., oligohydramnios) should also be examined.

Animals↗
PubMed1991

The risk of ligation of the left renal vein in resection of the abdominal aortic aneurysm.

The left renal vein can be ligated during aortic operation to attain better access to the perirenal aorta. This maneuver has been considered safe, with a low incidence of postoperative renal complications. This study was done to evaluate the risk of left renal vein ligation and its influence on renal function in patients with elective resection of abdominal aortic aneurysms. The records of 332 patients undergoing elective repair of abdominal aortic aneurysms during a five year period were reviewed. The clinical and operative data of patients who had left renal vein ligation and those who did not were similar. Left renal vein ligation was not associated with an increased mortality rate but was strongly associated with an increase in serum creatinine level and a clinical diagnosis of postoperative azotemia. Nine of 13 patients who had left renal vein ligation had postoperative azotemia compared with 21 of 319 patients without left renal vein ligation (p less than 0.001). The mean change of preoperative and postoperative creatinine value was an increase of 1.92 milligrams per deciliter in patients with left renal vein ligation in contrast with 0.26 milligram per deciliter in patients without ligation (p less than 0.00075). Thus, ligation of the left renal vein increases the risk of postoperative renal complications and its use should be selective.

Adult↗
PubMed1994

Renin and angiotensinogen mRNA expression in the kidneys of rats subjected to long-term bile duct ligation.

Activation of antinatriuretic systems such as the renin-angiotensin system, is of major importance in the pathogenesis of sodium retention in cirrhosis. In this study, we studied the intrarenal renin-angiotensin system by measuring renin and angiotensinogen mRNA expression in the kidney of rats subjected to long-term bile duct ligation in a phase before the development of ascites, when sodium retention is already present. Experiments were performed in sham-operated and bile duct-ligated rats 3 wk after surgery. Balance studies showed lower sodium excretion and greater sodium retention in the bile duct-ligated rats compared with the control animals. Plasma renin activity (4.41 +/- 1.01 ng Angiotensin I/ml/hr in the bile duct-ligated group vs. 4.20 +/- 0.74 in the controls) and plasma renin concentration were not different between the two groups. However, plasma renin substrate was significantly decreased in bile duct-ligated animals. Total kidney renin mRNA was significantly higher in the bile duct-ligated animals (0.83 +/- 0.14 densitometric units vs. 0.44 +/- 0.04 in the controls), as determined on Northern-blot analysis and densitometric quantitation. Angiotensinogen mRNA expression in the kidneys of bile duct-ligated rats was significantly decreased (0.09 +/- 0.01 densitometric units) compared with that of the controls (0.21 +/- 0.03). These results indicate that sodium-retaining, nonascitic bile duct-ligated rats show abnormalities of the intrarenal renin angiotensin system that precede changes in plasma renin activity. Our data suggest that the intrarenal renin angiotensin system may participate in the initiation of the renal pathophysiological abnormalities present in bile duct-ligated rats.

Angiotensinogen↗
PubMed1993

Single-site modifications of half-ligated hemoglobin reveal autonomous dimer cooperativity within a quaternary T tetramer.

The patterns of energetic response elicited by single-site hemoglobin mutations and chemical modifications have been determined in order to probe the dimer-dimer interface of the half-ligated tetramer (species [21]) that was previously shown to behave as allosterically distinct from both the unligated and fully ligated molecules. In this study the free energies of quaternary assembly (dimers to tetramers) were determined for a series of 24 tetrameric species in which one dimeric half-molecule is ligated (cyanomet hemes) while the adjacent alpha beta dimer is unligated and contains a single amino acid modification. Assembly energies have also been determined for tetramers bearing the same amino acid modifications but where the hemesites were completely vacant and additionally where they were fully occupied. A total of 72 molecular species were thus characterized. It was found that mutationally induced perturbations to the free energy of quaternary assembly were identical for the half-ligated tetramers and the unligated tetramers over the entire spatial distribution of altered sites, but exhibited a radically different pattern from that of the fully ligated molecules. These results indicate that the dimer-dimer interface of the half-ligated tetramer (species [21]) has the same quaternary structure as that of the unligated molecule, i.e., "quaternary T." This quaternary structure assignment of species [21] strongly supports the operation of a Symmetry Rule which translates changes in hemesite ligation into six T-->R quaternary switchpoints. Analysis of the observed Symmetry Rule behavior in relation to the measured distribution of cooperative free energies for the partially ligated species reveals significant cooperativity between alpha and beta subunits of the dimeric half-tetramer within quaternary T. The mutational results indicate that these interactions are not "paid for" by breaking or making noncovalent bonds at the dimer-dimer interface (alpha 1 beta 2). They arise from structural and energetic changes that are "internal" to the ligated dimer even though its association with the unligated dimer is required for the cooperativity to occur. Free energy of "tertiary constraint" is thus generated by the first binding step and is propagated to the second hemesite while the dimer-dimer interface alpha 1 beta 2 serves as a constraint. The "sequential" cooperativity that occurs within the half-molecule is thus preconditioned by the constraint of a quaternary T interface; release of this constraint by dissociation produces only noncooperative dimers.(ABSTRACT TRUNCATED AT 400 WORDS)

Allosteric Regulation↗
PubMed2006

A comparison of different ligation methods on friction.

INTRODUCTION: An elastomeric module with a polymeric coating has been developed to reduce the friction of sliding mechanics. This in-vitro study examined the stability of the coating and compared the frictional properties of coated modules with those of other common ligation methods. METHODS: Six ligation methods (regular uncoated, slick [coated], conventional silver, easy-to-tie, silicone-impregnated, and standard silver modules) were used with standard stainless steel brackets and 0.019 x 0.025-in archwires, and resistance to movement was measured. Two self-ligating (Speed [Strite Industries, Cambridge, Ontario, Canada] and Damon 2 [Sybron Dental Specialities Ormco, Orange, Calif]) brackets were also tested. RESULTS: The Damon 2 self-ligating brackets produced less friction than the other ligation methods, followed by the coated modules. There was no significant difference between the frictional resistance of brackets ligated with regular uncoated, silicone-impregnated, and easy-to-tie modules. Speed self-ligating brackets produced less friction than regular uncoated, conventional silver, and standard silver modules. The frictional properties of coated modules were not significantly affected by repeating the test 5 times or by storage in saliva for a week. CONCLUSIONS: Damon 2 brackets produced no recordable friction of ligation. Coated modules produced 50% less friction than all other ligation methods except Damon 2. The coating was resistant to the simulated effects of the oral environment. Different methods of human saliva application were found to affect the frictional properties of the coating.

Analysis of Variance↗
PubMed2003

Optimization and generality of a small deoxyribozyme that ligates RNA.

In vitro evolution was previously used to identify a small deoxyribozyme, 7Q10, that ligates RNA with formation of a 2'-5' phosphodiester linkage from a 2',3'-cyclic phosphate and a 5'-hydroxyl group. Ligation occurs in a convenient "binding arms" format analogous to that of the well-known 10-23 and 8-17 RNA-cleaving deoxyribozymes. Here, we report the optimization and generality of 7Q10 as a 2'-5' RNA ligase. By comprehensive mutagenesis of its 16-nucleotide enzyme region, the parent 7Q10 sequence is shown to be optimal for RNA ligation yield, although several mutations are capable of increasing the ligation rate approximately fivefold at the expense of yield. The 7Q10 deoxyribozyme ligates any RNA substrates that form the sequence motif UA GR (arrowhead=ligation site and R=purine), providing at least 30% yield of ligated RNA in approximately 1-2 hours at 37 degrees C and pH 9.0. Comparable yields are obtained in approximately 12-24 hours at pH 7.5, which may be more suitable for larger RNAs that are more sensitive to non-specific degradation. For RNA substrates that form the related ligation junction UA GY (Y=pyrimidine), somewhat lower yields are obtained, but significant ligation activity is still observed. These data establish that 7Q10 is a generally applicable RNA ligase. A plot of log(k(obs)) versus pH from pH 6.9 to 9.0 has a slope of just under 1, suggesting that a single deprotonation occurs during the rate-determining reaction step. The compact 7Q10 deoxyribozyme has both practical utility and the potential for increasing our structural and mechanistic understanding of how nucleic acids can mediate chemical reactions.

Base Sequence↗
PubMed1992

Cooperative oxygen binding, subunit assembly, and sulfhydryl reaction kinetics of the eight cyanomet intermediate ligation states of human hemoglobin.

Correlations between the energetics of cooperativity and quaternary structural probes have recently been made for the intermediate ligation states of Hb [Daugherty et al. (1991) Proc. Natl. Acad. Sci. US 88, 1110-1114]. This has led to a "molecular code" which translates configurations of the 10 ligation states into switch points of quaternary transition according to a "symmetry rule"; T-->R quaternary structure change is governed by the presence of at least one heme-site ligand on each of the alpha beta dimeric half-molecules within the tetramer [see Ackers et al. (1992) Science 255, 54-63, for summary]. In order to further explore this and other features of the cooperative mechanism, we have used oxygen binding to probe the energetics and cooperativities for the vacant sites of the cyanomet ligation species. We have also probed structural aspects of all eight cyanomet ligation intermediates by means of sulfhydryl reaction kinetics. Our oxygen binding results, obtained from a combination of direct and indirect methods, demonstrate the same combinatorial aspect to cooperativity that is predicted by the symmetry rule. Overall oxygen affinities of the two singly-ligated species (alpha +CN beta)(alpha beta) and (alpha beta +CN)(alpha beta) were found to be identical (pmedian = 2.4 Torr). In contrast, the doubly-ligated species exhibited two distinct patterns of oxygen equilibria: the asymmetric species (alpha +CN beta +CN)(alpha beta) showed very high cooperativity (nmax = 1.94) and low affinity (pmedian = 6.0 Torr), while the other three doubly-ligated species showed diminished cooperativity (nmax = 1.23) and considerably higher oxygen affinity (pmedian = 0.4 Torr). Extremely high oxygen affinities were found for the triply-ligated species (alpha +CN beta +CN)(alpha beta +CN) and (alpha +CN beta +CN)(alpha +CN beta) (pmedian = 0.2 Torr). Their oxygen binding free energies are considerably more favorable than those of the alpha and beta subunits within the dissociated alpha beta dimer, demonstrating directly the quaternary enhancement effect, i.e., enhanced oxygen affinity at the last binding step of tetramer relative to the dissociated protomers. Oxygen binding free energies measured for the alpha subunit within the isolated (alpha beta +CN) dimer and for the beta subunit within the isolated (alpha +CN beta) dimer sum to the free energy for binding two oxygens to normal hemoglobin dimers (-16.3 +/- 0.2 versus -16.7 +/- 0.2, respectively), arguing against cooperativity in the isolated dimer. Correlations were established between cooperative free energies of the 10 cyanomet ligation microstates and the kinetics for reacting their free sulfhydryl groups.(ABSTRACT TRUNCATED AT 400 WORDS)

Chromatography, Gel↗
PubMed1996

ATPase activity of Escherichia coli Rep helicase is dramatically dependent on DNA ligation and protein oligomeric states.

The Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA using the energy derived from ATP binding and hydrolysis. Rep functions as a dimer but assembles to its active dimeric form only on binding DNA. Each promoter of a dimer contains a DNA binding site that can bind either single-stranded (S) or duplex (D) DNA. The dimer can bind up to two oligodeoxynucleotides in five DNA-ligation states: two half-ligated states, P2S and P2D, and three fully-ligated states, P2S2, P2D2, and P2SD. We have previously shown that the relative stabilities of these ligation states are allosterically regulated by the binding and hydrolysis of ATP and have proposed an "active rolling" model for DNA unwinding where the enzyme cycles through a series of these ligation states in a process that is coupled to the catalytic cycle of ATP hydrolysis [Wong, I., & Lohman, T.M., (1992), Science 256, 350-355]. THe basal ATPase activity of Rep protein is stimulated by ss DNA binding and by protein dimerization. We have measured the steady-state ATPase activities of Rep bound to dT(pT)15 in each distinct ss DNA ligation state (PS, P2S, and P2S2) to compare with our previous measurements with unligated Rep monomer (P) [Moore, K.J.M., & Lohman, T.M. (1994) Biochemistry 33, 14550]. We find the ATPase activity of Rep is influenced dramatically by both dimerization and ss DNA ligation state, with the following kcat values for ATP hydrolysis increasing by over 4 orders of magnitude: 2.1 x 10(-3) s(-1) for P, 2.17 +/- 0.04 s(-1) for PS, 16.5 +/- 0.2 s(-1) for P2S, and 71 +/- 2.5 s(-1) for P2S2 (20 mM Tris-HCl, pH 7.5, 6mM NaCl, 5 mM MgCl2, 10% glycerol, 4 degrees C). The apparent KM's for ATP hydrolysis are 2.05 +/- 0.1 microM for PS and 2.7 +/- 0.2 microM for P2S. These widely different ATPase activities reflect the allosteric effects of DNA ligation and demonstrate that cooperative communication occurs between the ATP and DNA site of both subunits of the Rep dimer. These results further emphasize the need to explicitly consider the population distribution of oligomerization and DNA ligation states of the helicase when attempting to infer information about elementary processes such as helicase translocation based solely on macroscopic steady-state ATPase measurements.

Adenosine Triphosphatases↗