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At least 577 records · Page 32Linked to original sources
PubMed2000

The delta subunit of DNA polymerase III holoenzyme serves as a sliding clamp unloader in Escherichia coli.

In Escherichia coli, the circular beta sliding clamp facilitates processive DNA replication by tethering the polymerase to primer-template DNA. When synthesis is complete, polymerase dissociates from beta and DNA and cycles to a new start site, a primed template loaded with beta. DNA polymerase cycles frequently during lagging strand replication while synthesizing 1-2-kilobase Okazaki fragments. The clamps left behind remain stable on DNA (t(12) approximately 115 min) and must be removed rapidly for reuse at numerous primed sites on the lagging strand. Here we show that delta, a single subunit of DNA polymerase III holoenzyme, opens beta and slips it off DNA (k(unloading) = 0.011 s(-)(1)) at a rate similar to that of the multisubunit gamma complex clamp loader by itself (0.015 s(-)(1)) or within polymerase (pol) III* (0.0065 s(-)(1)). Moreover, unlike gamma complex and pol III*, delta does not require ATP to catalyze clamp unloading. Quantitation of gamma complex subunits (gamma, delta, delta', chi, psi) in E. coli cells reveals an excess of delta, free from gamma complex and pol III*. Since pol III* and gamma complex occur in much lower quantities and perform several DNA metabolic functions in replication and repair, the delta subunit probably aids beta clamp recycling during DNA replication.

Blotting, Western↗
PubMed2001

Mechanism of beta clamp opening by the delta subunit of Escherichia coli DNA polymerase III holoenzyme.

The beta sliding clamp encircles the primer-template and tethers DNA polymerase III holoenzyme to DNA for processive replication of the Escherichia coli genome. The clamp is formed via hydrophobic and ionic interactions between two semicircular beta monomers. This report demonstrates that the beta dimer is a stable closed ring and is not monomerized when the gamma complex clamp loader (gamma(3)delta(1)delta(1)chi(1)psi(1)) assembles the beta ring around DNA. delta is the subunit of the gamma complex that binds beta and opens the ring; it also does not appear to monomerize beta. Point mutations were introduced at the beta dimer interface to test its structural integrity and gain insight into its interaction with delta. Mutation of two residues at the dimer interface of beta, I272A/L273A, yields a stable beta monomer. We find that delta binds the beta monomer mutant at least 50-fold tighter than the beta dimer. These findings suggest that when delta interacts with the beta clamp, it binds one beta subunit with high affinity and utilizes some of that binding energy to perform work on the dimeric clamp, probably cracking one dimer interface open.

Adenosine Triphosphate↗
PubMed2003

Mechanism of the delta wrench in opening the beta sliding clamp.

The beta sliding clamp encircles DNA and tethers DNA polymerase III holoenzyme to the template for high processivity. The clamp loader, gamma complex (gamma 3 delta delta'chi psi), assembles beta around DNA in an ATP-fueled reaction. The delta subunit of the clamp loader opens the beta ring and is referred to as the wrench; ATP modulates contact between beta and delta among other functions. Crystal structures of delta.beta and the gamma 3 delta delta' minimal clamp loader make predictions of the clamp loader mechanism, which are tested in this report by mutagenesis. The delta wrench contacts beta at two sites. One site is at the beta dimer interface, where delta appears to distort the interface by via a steric clash between a helix on delta and a loop near the beta interface. The energy for this steric clash is thought to derive from the other site of interaction, in which delta binds to a hydrophobic pocket in beta. The current study demonstrates that rather than a simple steric clash with beta, delta specifically contacts beta at this site, but not through amino acid side chains, and thus is presumably mediated by peptide backbone atoms. The results also imply that the interaction of delta at the hydrophobic site on beta contributes to destabilization of the beta dimer interface rather than acting solely as a grip of delta on beta. Within the gamma complex, delta' is proposed to prevent delta from binding to beta in the absence of ATP. This report demonstrates that one or more gamma subunits also contribute to this role. The results also indicate that delta' acts as a backboard upon which the gamma subunits push to attain the ATP induced change needed for the delta wrench to bind and open the beta ring.

Adenosine Triphosphate↗
PubMed2005

The 9-1-1 checkpoint clamp physically interacts with polzeta and is partially required for spontaneous polzeta-dependent mutagenesis in Saccharomyces cerevisiae.

The use of translesion synthesis (TLS) polymerases to bypass DNA lesions during replication constitutes an important mechanism to restart blocked/stalled DNA replication forks. Because TLS polymerases generally have low fidelity on undamaged DNA, the cell must regulate the interaction of TLS polymerases with damaged versus undamaged DNA to maintain genome integrity. The Saccharomyces cerevisiae checkpoint proteins Ddc1, Rad17, and Mec3 form a clamp-like structure (the 9-1-1 clamp) that has physical similarity to the homotrimeric sliding clamp proliferating cell nuclear antigen, which interacts with and promotes the processivity of the replicative DNA polymerases. In this work, we demonstrate both an in vivo and in vitro physical interaction between the Mec3 and Ddc1 subunits of the 9-1-1 clamp and the Rev7 subunit of the Polzeta TLS polymerase. In addition, we demonstrate that loss of Mec3, Ddc1, or Rad17 results in a decrease in Polzeta-dependent spontaneous mutagenesis. These results suggest that, in addition to its checkpoint signaling role, the 9-1-1 clamp may physically regulate Polzeta-dependent mutagenesis by controlling the access of Polzeta to damaged DNA.

Base Sequence↗
PubMed1999

The behaviour pattern of lambs after castration using a rubber ring and/or castrating clamp with or without local anaesthetic.

AIMS: To determine if individual behaviours may be used to compare the distress caused by the different methods of castration. METHODS: A controlled experiment in which the behaviour pattern of 174 lambs given one of twenty treatments (six ring, five clamp plus ring and four clamp castration with a variety of local anaesthetic placements plus five control treatments) were monitored for 4 hours from about 5 seconds after treatment. RESULTS: The contrast between the behaviour patterns of lambs castrated by different methods with or without local anaesthesia and control groups allowed the identification of individual behaviours stimulated by the different methods of castration. After ring castration there was an increase in restlessness, in contrast to clamp castration, which had no obvious individual behaviour associated with it during the 4 hours after treatment. Clamp plus ring castration resulted in a behaviour pattern similar to those caused by ring castration alone. CONCLUSIONS: Individual behaviours can only be used to compare the distress caused by different methods of castration if the methods stimulate similar behaviours. The distress which occurs after ring or clamp castration cannot be compared using behavioural observations because the two techniques stimulate different types of behaviour.

Journal Article↗
PubMed1998

Femoral artery haemostasis with a pneumatic compression device versus a clamp after coronary angiography.

To evaluate the safety and efficacy of a new pneumatic compression device for achieving haemostasis after femoral artery catheterization, 1,017 patients undergoing selective coronary angiography by a 5F unilateral femoral route were prospectively randomised to pneumatic or the routinely used clamp compression technique. All initial bleedings could be controlled in the pneumatic group, whereas in 38 patients (8%) of the clamp group the initial positioning of the clamp was unsuccessful or was not tolerated by the patient (p < 0.05). Ultrasound Doppler study of the puncture site because of suspected postcatheterization vascular complication revealed two haematomas which needed no further measure and two pseudoaneurysms which were successfully treated with ultrasound-guided compression or surgical repair. The rate of complications requiring treatment (pseudoaneurysms) was 0.2% overall, 0.5% in the clamp group and nil in the pneumatic compression group (NS). We conclude that the pneumatic compression device is effective, convenient and at least as safe as the clamp and, by shortening the time in the catheterization laboratory, offers time for further angiograms.

Aged↗
PubMed1976

An improved vaseline gap voltage clamp for skeletal muscle fibers.

A Vaseline gap potentiometric recording and voltage clamp method is developed for frog skeletal muscle fibers. The method is based on the Frankenhaeuser-Dodge voltage clamp for myelinated nerve with modifications to improve the frequency response, to compensate for external series resistance, and to compensate for the complex impedance of the current-passing pathway. Fragments of single muscle fibers are plucked from the semitendinosus muscle and mounted while depolarized by a solution like CsF. After Vaseline seals are formed between fluid pools, the fiber ends are cut once again, the central region is rinsed with Ringer solution, and the feedback amplifiers are turned on. Errors in the potential and current records are assessed by direct measurements with microelectrodes. The passive properties of the preparation are simulated by the "disk" equivalent circuit for the transverse tubular system and the derived parameters are similar to previous measurements with microelectrodes. Action potentials at 5 degrees C are long because of the absence of delayed rectification. Their shape is approximately simulated by solving the disk model with sodium permeability in the surface and tubular membranes. Voltage clamp currents consist primarily of capacity currents and sodium currents. The peak inward sodium current density at 5 degrees C is 3.7 mA/cm2. At 5 degrees C the sodium currents are smoothly graded with increasing depolarization and free of notches suggesting good control of the surface membrane. At higher temperatures a small, late extra inward current appears for small depolarizations that has the properties expected for excitation in the transverse tubular system. Comparison of recorded currents with simulations shows that while the transverse tubular system has regenerative sodium currents, they are too small to make important errors in the total current recorded at the surface under voltage clamp at low temperature. The tubules are definitely not under voltage clamp control.

Action Potentials↗
PubMed1997

Delayed clamping of the umbilical cord improves hematologic status of Guatemalan infants at 2 mo of age.

Iron deficiency anemia is a serious health problem that affects the physical and cognitive development of children. Therefore, it is important to develop cost-effective interventions to improve the hematologic status of the millions of children affected by this condition worldwide. We studied 69 Guatemalan infants who had been randomly assigned to one of three groups at the time of delivery: 1) cord clamping immediately after delivery (n = 21); 2) clamping when the cord stopped pulsating, with the infant placed at the level of the placenta (n = 26); or 3) clamping when the cord stopped pulsating, with the newborn placed below the level of the placenta (n = 22). Maternal and infant hematologic assessments were performed at the time of delivery and 2 mo postpartum. At baseline the groups had similar socioeconomic, demographic, and biomedical characteristics and the newborns had similar hematocrit status. Two months after delivery, infants in the two groups with delayed cord clamping had significantly higher hematocrit values and hemoglobin concentrations than did those in the early-clamping group. The percentage with hematocrit values < 0.33 was 88% in the control group compared with 42% in group 2 and 55% in group 3 (P = 0.01). These results suggest that waiting until the umbilical cord stops pulsating (approximately 1 min after delivery) is a feasible low-cost intervention that can reduce anemia in infants in developing countries.

Adult↗
PubMed2001

Effects of portal triad clamping on haemodynamic conditions during laparoscopic liver resection.

To evaluate the haemodynamic effects of portal triad clamping (PTC) during laparoscopic liver resection, 10 patients without cardiac disease were studied by invasive monitoring including a pulmonary artery catheter and were compared with a control group of 10 patients undergoing liver resection by laparotomy. During laparoscopic surgery, intra-abdominal pressure was kept below 14 mm Hg and minute ventilation was adjusted to prevent hypercapnia. Measurements were made before PTC (T1), 5 min after PTC (T2) and 5 min after clamp release (T3). During clamping with pneumoperitoneum, mean arterial pressure (MAP) remained stable (+2%; not significant), systemic vascular resistance (SVR) increased by 37% (P<0.01, T2 vs T1) and cardiac index (CI) decreased by 19% (P<0.01, T2 vs T1). During laparotomy and clamping, MAP increased by 18% (P<0.01, T2 vs T1), SVR increased by 36% (P<0.01, T2 vs T1) and CI decreased by 9% (not significant). We were unable to demonstrate a difference in haemodynamic changes during clamping with pneumoperitoneum vs the open surgical technique, but in a small number of patients this lack of difference could have been a result of inadequate statistical power. The haemodynamic changes that we found were well tolerated in these patients, who had normal cardiac function.

Adult↗
PubMed1985

Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis.

Duplex DNA fragments differing by single base substitutions can be separated by electrophoresis in denaturing gradient polyacrylamide gels, but only substitutions in a restricted part of the molecule lead to a separation (1). In an effort to circumvent this problem, we demonstrated that the melting properties and electrophoretic behavior of a 135 base pair DNA fragment containing a beta-globin promoter are changed by attaching a GC-rich sequence, called a 'GC-clamp' (2). We predicted that these changes should make it possible to resolve most, if not all, single base substitutions within fragments attached to the clamp. To test this possibility we examined the effect of several different single base substitutions on the electrophoretic behavior of the beta-globin promoter fragment in denaturing gradient gels. We find that the GC-clamp allows the separation of fragments containing substitutions throughout the promoter fragment. Many of these substitutions do not lead to a separation when the fragment is not attached to the clamp. Theoretical calculations and analysis of a large number of different mutations indicate that approximately 95% of all possible single base substitutions should be separable when attached to a GC-clamp.

Animals↗
PubMed2003

Steric inhibition of human immunodeficiency virus type-1 Tat-dependent trans-activation in vitro and in cells by oligonucleotides containing 2'-O-methyl G-clamp ribonucleoside analogues.

We report the synthesis of a novel 2'-O-methyl (OMe) riboside phosphoramidite derivative of the G-clamp tricyclic base and incorporation into a series of small steric blocking OMe oligonucleotides targeting the apical stem-loop region of human immunodeficiency virus type 1 (HIV-1) trans- activation-responsive (TAR) RNA. Binding to TAR RNA is substantially enhanced for certain single site substitutions in the centre of the oligonucleotide, and doubly substituted anti-TAR OMe 9mers or 12mers exhibit remarkably low binding constants of <0.1 nM. G-clamp-containing oligomers achieved 50% inhibition of Tat-dependent in vitro transcription at approximately 25 nM, 4-fold lower than for a TAR 12mer OMe oligonucleotide and better than found for any other oligonucleotide tested to date. Addition of one or two OMe G-clamps did not impart cellular trans-activation inhibition activity to cellularly inactive OMe oligonucleotides. Addition of an OMe G-clamp to a 12mer OMe-locked nucleic acid chimera maintained, but did not enhance, inhibition of Tat-dependent in vitro transcription and cellular trans-activation in HeLa cells. The results demonstrate clearly that an OMe G-clamp has remarkable RNA-binding enhancement ability, but that oligonucleotide effectiveness in steric block inhibition of Tat-dependent trans-activation both in vitro and in cells is governed by factors more complex than RNA-binding strength alone.

Anti-HIV Agents↗
PubMed2006

Fluorescence detection of 8-oxoguanosine by G-clamp derivatives.

8-Oxoguanosine, which is derived by the oxidation of guanosine (dG), is known to induce transversion mutations (G:C-->T:A) in DNA. The compounds with small molecular weight for recognizing 8-oxoG were designed on the basis of the structure of the G-clamp, which is reported to have selective affinity toward guanosine. The G-clamp derivatives with the additional binding units toward 8-oxoG were effectively synthesized and named "8-oxoG-clamps". Binding properties were measured by fluorescence quenching of 8-oxoG-clamps. Among the synthesized derivatives, 8-oxoG-clamp with N-benzyloxycarbonyl (Cbz) unit completely discriminated 8-oxoG from other nucleosides by fluorescence quenching.

Carbamates↗
PubMed2006

Clamping of the dialysate outlet line in the Genius dialysis system does not alter dialysate flow or clearances.

BACKGROUND: The Genius dialysis system is advocated as a tool to perform slow extended daily dialysis (SLEDD) in patients with acute renal failure at the intensive care unit. At low blood flows however, it is sometimes problematic to maintain sufficient systemic pressure in the dialysate circuit, a problem that can be overcome by clamping the dialysate outflow line. This intervention can however decrease the flow in the dialysate circuit, and can thus potentially decrease the clearance. This article analyses the impact of this intervention on the blood and dialysate flows and on the removal of retention products. METHODS: The study was done in 20 stable chronic dialysis patients to avoid additional bias caused by changing comorbidities in ICU patients. Patients were treated by Genius, once with clamping and once without clamping, cross-over in a randomized fashion, and with 1 week interval. Flows in the dialysate and blood circuit were measured with a transsonic flow probe. Urea, creatinine, Beta 2 microglobulin and phosphate were measured in the dialysate, and in the serum before and after dialysis. RESULTS: There was no clinically significant difference in blood or dialysate flows, nor in clearance or removal of retention products during the sessions with or without clamping. CONCLUSIONS: The technique of using clamping of the dialysate outflow line in the Genius system to increase systemic pressure, when the system is used in SLEDD, is a safe technique which does not alter the clearances.

Aged↗
PubMed1984

Effects of infrarenal aortic cross-clamping on renal hemodynamics in humans.

While the systemic cardiovascular consequences of infrarenal aortic cross-clamping during aortic abdominal surgery are well documented, its repercussions on renal hemodynamics in humans have not been reported. In 12 patients, scheduled for elective aortic surgery, renal clearances, using 51Cr EDTA and 125I hippuran, were measured before, during, and after infrarenal aortic cross-clamping. A continuous infusion of mannitol 20% at a rate of 100 ml/h was administered throughout the study. Arterial and renal venous blood sampling, obtained at the midpoint of each period, permitted calculation of the extraction fraction of 125I hippuran and accurate determination of renal blood flow and its cortical-extracortical distribution. Although cardiac output and systemic vascular resistance did not change significantly between the three study periods, infrarenal aortic cross-clamping decreased 125I hippuran clearance by 29 +/- 15% (P less than 0.05) and renal blood flow by 38 +/- 14% (P less than 0.001). Simultaneously, an increase of 75 +/- 31% in renal vascular resistance (P less than 0.05) was observed and the extraction fraction of 125I hippuran increased from 0.67 +/- 0.05 to 0.74 +/- 0.05 (P less than 0.01). All of these changes, which indicate global diminution of renal perfusion with a redistribution of renal blood flow toward the cortical compartment, persisted for at least 1 h after release of the aortic clamp. Early signs of renal tubular damage, such as the appearance of lysozyme and ligandine in the urine, however, were never observed. The authors conclude that infrarenal aortic cross-clamping produces profound and sustained alterations in renal hemodynamics and may be harmful in patients with impaired renal function or when surgical occlusion of the aorta is prolonged.

Aged↗
PubMed1992

Treatment of proximal aortic hypertension after thoracic aortic cross-clamping in dogs. Phlebotomy versus sodium nitroprusside/isoflurane.

Thoracic aortic cross-clamping causes proximal aortic hypertension. Theoretically, the method used to treat hypertension can influence spinal cord perfusion pressure and neurologic outcome. Phlebotomy was compared to sodium nitroprusside/isoflurane in terms of ability to treat increased proximal mean aortic pressure (MAPp) after thoracic aortic cross-clamping in dogs. Dogs were assigned randomly to one of three groups depending on the method used to treat hypertension after cross clamping: 1) phlebotomy (n = 10); 2) sodium nitroprusside/isoflurane (n = 11); and 3) control (no treatment) (n = 8). In each dog, anesthesia was maintained with isoflurane in oxygen, 1.4% end-tidal. The thoracic aorta was occluded 2.5 cm distal to the left subclavian artery for 50 min and then was released. Hemodynamics, cerebrospinal fluid pressure (CSFP), and regional blood flows by the radioactive microsphere technique, were measured at 1) baseline; 2) 2 min after aortic cross-clamping; 3) after treatment of proximal aortic hypertension; 4) 5 min after aortic unclamping; and 5) 30 min after resuscitation. At 24 h, a neurologic assessment was performed. Thoracic aortic cross-clamping increased MAPp, decreased distal MAP (MAPd), and reduced lumbar spinal cord perfusion pressure (SCPPl), [SCPPl = MAPd - CSFP], in all three groups. Control of increased MAPp necessitated removal of 36 +/- 9 ml/kg of blood in the phlebotomy group. In the sodium nitroprusside/isoflurane group, sodium nitroprusside (16 micrograms.kg-1.min-1) was infused and end-tidal isoflurane concentration increased to 2.5 +/- 0.7%, restoring MAPp to baseline level.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

The effects of thoracic aortic cross-clamping and declamping on visceral organ blood flow.

Blood flow was measured using radioactive microspheres in 11 macaque monkeys 1) before hemorrhage shock, 2) after onset of shock, 3) after aortic cross-clamping and resuscitation, and 4) after release of the cross-clamp and stabilization. Hemodynamic parameters (cardiac output, arterial, right atrial and left atrial pressure) and blood gases were also monitored. Total abdominal organ flow fell with hemorrhage and fell further with aortic clamping. Reinfusion of shed volume did not restore abdominal organ flow (4.7% baselines) but increased LAP and cardiac output to the upper body. Release of the cross-clamp produced profound acidosis that was treated effectively with NcHCO3. After stabilization of blood, flow to kidney remained low (49% baseline) although intestinal flow was increased threefold (320% of baseline). It is clear that thoracic aortic cross-clamping in shock further compromises already reduced visceral blood flow and may contribute to the problem of ischemic multiple organ failure after resuscitation from hemorrhagic shock.

Abdomen↗
PubMed1987

Experimental reclamping of free-flap pedicles: the effect of prolonged stasis on the anastomoses and clamp sites.

The effect of static blood in direct contact with areas of microvascular anastomoses and previous clamp application for prolonged periods of time has been investigated. The free groin flap was used as a model in 27 white rabbits. The flap pedicle vessels were reclamped proximal to the anastomoses and areas of previous clamp application for periods of time varying between 30 minutes and 4 hours after 15 minutes of blood flow over these areas. A 100 percent patency rate was achieved despite the long periods of reclamping. Histologically, minor intimal damage was visible in the immediate period following anastomoses and clamping of the vessels. After 2 weeks, despite a thickened myofibroblastic intimal lesion, an intact endothelial layer was observed. No evidence of thrombosis could be demonstrated in either period. We postulate that vessels carefully treated and with technically well-performed anastomoses can be regarded as "normal" vessels after 15 minutes of blood flow over the anastomoses and clamp sites. We suggest that when required, microvascular clamps may then be reapplied without risk for prolonged periods of time despite static blood being in contact with these areas.

Animals↗
PubMed2000

Modified towel-clamp technique to effect reduction of displaced mandible fractures.

We report a technique that uses a modified standard towel clamp, allowing a single surgeon to perform and maintain an anatomic reduction of displaced mandible fractures simultaneous with the application of internal fixation. The reduced convergent angle of the modified towel clamp allows bicortical engagement of the clamp, which prevents comminution of the fracture or the outer cortex of the mandible. Additionally, the modification allows the clamp to engage the bone with less exposure than conventional towel clamps. In our clinical experience of treating more than 100 mandible fractures a year, this technique proves superior to others described in the literature.

Humans↗