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At least 1,135 records · Page 63Linked to original sources
PubMed1992

Purification and patch clamp analysis of a 40-pS channel from rat liver mitochondria.

Patch clamp analysis of membranes reconstituted with a fraction isolated from detergent-solubilized mitochondrial membranes by affinity chromatography on immobilized quinine earlier indicated the presence of two classes of ion channels, of about 40- and 140-pS conductance in medium including 150 mM KCl. Now a 57-kDa constituent of the quinine-affinity column eluate has been identified as the 40-pS channel. Protein fractions derived from the quinine-affinity column eluate by preparative isoelectric focusing with a Rotofor cell have been reconstituted into phospholipid vesicle membranes by detergent dialysis, and vesicles have been enlarged for patch clamping by dehydration and rehydration. Voltage clamp analysis has been carried out on excised patches bathed symmetrically in buffered medium containing 150 mM KCl and 100 microM CaCl2. Patches of membrane incorporating the 57-kDa protein exhibit 40-pS conductance transitions. The magnitude of conductance transitions is similar when Na+ replaces K+ in the bathing medium, indicating little selectivity of the 40-pS channel for K+ relative to Na+. Another fraction derived from the quinine-affinity column eluate is found to contain the larger channel, now estimated to have an average conductance of about 130 pS. Patches of control membrane prepared in the same way but without protein exhibit no channel activity.

Animals↗
PubMed2004

On the solution structure of the T4 sliding clamp (gp45).

Examination by time-resolved fluorescence spectroscopy of the trimeric bacteriophage T4 clamp protein labeled across its three subunit interfaces with a fluorescence resonance energy transfer (FRET) pair indicates that the clamp exists in just one state in solution, with one open and two closed interfaces. This is in contrast to what is observed in the X-ray crystal structure. The population distribution of the trFRET distance is bimodal, giving 67% as 17 A and 33% as 42 A. This leads to the conclusion that gp45 exists in an asymmetric open state in solution. The further increase in the separation of the FRET pair in the presence of the clamp loader and ATP may be ascribed to either further opening of the open interface or the opening of a closed interface. The ramifications for replisome remodeling by this pathway are discussed.

Bacteriophage T4↗
PubMed1999

Sliding clamp of the bacteriophage T4 polymerase has open and closed subunit interfaces in solution.

The sliding clamps of bacteriophage T4 (gp45), Escherichia coli (beta clamp), and yeast (PCNA) are required for processive DNA synthesis by their cognate DNA polymerases. The X-ray crystal structures of all three of these clamps have been shown to be closed, circular complexes. This paper reports investigations of the solution structure of bacteriophage T4 gp45 by analytical ultracentrifugation, fluorescence, and hydrodynamic modeling. Mutants of gp45 with inter- and intrasubunit disulfide bonds were created to alter the solution structure of gp45, with additional mutagenesis used to investigate the importance of the proline-rich loop region found between the two domains of each gp45 monomer. The wild-type gp45 trimer assembles from monomers cooperatively with a dissociation constant of 0.21 microM2 and values between 0.088 and 0. 32 microM2 for the mutants. Velocity ultracentrifugation experiments showed that wild-type gp45 possesses a sedimentation coefficient strongly dependent on concentration, typical of asymmetric or elongated molecules, that when extrapolated to zero concentration yields a sedimentation coefficient of 4.0 S. The loop and the disulfide mutants exhibited sedimentation coefficients with little concentration dependence, typical of symmetric or spherical molecules, that when extrapolated to zero concentration yielded sedimentation coefficients of 4.4-4.8 S. The lower sedimentation coefficient in the former case is consistent with wild-type gp45 being more asymmetric or elongated than the mutant forms. Fluorescence-resonance energy-transfer experiments were used to measure the distance between two amino acids (W91 and V162C-coumarin) on opposite sides of the gp45 subunit interface. For an intrasubunit disulfide mutant, the distance between these two amino acids was determined to be 19 A (14 A in the X-ray crystal structure), consistent with a closed complex. For the mutants without intrasubunit disulfides, the efficiency of fluorescence-resonance energy transfer was in accord with a model of gp45 being an open complex composed of two closed subunit interfaces and a third open interface separated by a distance of 35-38 A. The collective data supplemented with hydrodynamic modeling were consistent with gp45 subunit separation achieved within the plane of the gp45 ring.

Adenosine Triphosphatases↗
PubMed2002

Control of the diatropic pi ring current in strained benzenes: effects of annelation with cyclopropa, cyclobuta, and cyclobutadieno clamping groups.

Direct visualization of the pi current density maps of highly strained annelated benzenes containing cyclopropa, cyclobuta, and cyclobutadieno clamps, alone and in combination, using a reliable distributed-origin, coupled Hartree-Fock method, shows the robustness of the classical benzene diatropic pi ring current. When only saturated clamps are used, the benzene ring current is essentially unchanged. In contrast, annelation with one or more cyclobutadieno clamps disrupts the benzene ring current. Analysis of orbital contributions to the current density maps gives a unified account of these observations in terms of the nature of the HOMO-LUMO transition.

Journal Article↗
PubMed2006

Theoretical simulations of clamping levels in optical power limiting.

Multiphysics modeling, combining quantum mechanical and classical wave mechanical theories, of clamping levels has been performed for a platinum(II) organic compound in a sol-gel glass matrix. A clamping level of 2.5 microJ is found for a pulse duration of 10 ns. The excited-state absorption in the triplet manifold is shown to be crucial for clamping to occur.

Journal Article↗
PubMed2006

Convergent evolution of clamp-like binding sites in diverse chaperones.

Molecular chaperones have evolved diverse tertiary and quaternary structures to stabilize non-native polypeptides and facilitate their transition to the native state. Indeed, different families of chaperones lack sequence similarity, and few are represented ubiquitously in all three domains of life. Despite their discrete evolutionary paths, recent crystal structures reveal that many chaperones use seemingly convergent strategies to bind non-native proteins. This crystallographic evidence shows, or strongly suggests, that chaperones including prefoldin, Skp, trigger factor, Hsp40 and Hsp90 have clamp-like structural features used to grip substrate proteins. We explore the notion that clamp-like structures are evolutionarily favored by both ATP-dependent and ATP-independent molecular chaperones. Presumably, clamps present a multivalent binding surface ideal for protecting unstable protein conformers until they reach the native state or are transferred to another component of the folding machinery.

Adenosine Triphosphate↗
PubMed2001

Survival and extinction of delocalized ring currents in clamped benzenes.

Direct visualisation of induced current density in clamped benzenes 1-4 distinguishes between saturated clamping groups, for which the central benzene ring retains a conventional diamagnetic ring current, and strongly interacting, unsaturated clamps, for which the central ring supports only the localised circulations expected of a 1,3,5-cyclohexatriene with fully fixed double bonds.

Journal Article↗
PubMed2004

Which pi-clamped conjugated monocycles exhibit ring currents?

Quenching/survival of ring currents in pi-clamped conjugated monocycles is controlled by the match or mismatch in parity between the frontier orbitals of the central pi-conjugated 4n+2/4n monocycle and those of the clamps. Changes in ring current are not primarily caused by bond alternation or 'Mills-Nixon' effects; current and geometry changes on clamping are both consequences of electronic structure.

Journal Article↗
PubMed2001

The effect of the Lockit epidural catheter clamp on epidural migration: a controlled trial.

We investigated the efficacy of using the Lockit epidural catheter clamp in preventing epidural catheter migration in patients receiving postoperative analgesia via an epidural infusion after major surgery. Patients were randomly allocated to receive either a standard epidural dressing, a coiled catheter with transparent adhesive dressing and tape (n = 54), or the Lockit epidural catheter clamp (n = 48). There was no movement from the insertion position in 88% of the Lockit group compared with only 28% in the standard group (p < 0.001). Outward migration of > 2 cm occurred in 26% of the standard group compared with just 6% of the Lockit group (p < 0.01). Inward migration of > 1 cm occurred in 17% of the standard group but in none of the Lockit group (p < 0.01). Catheter migration was assessed as being the cause of analgesic failure in 15% of the standard group but in only 4% of the Lockit group (p < 0.05). We conclude that the Lockit epidural catheter clamp significantly reduces catheter migration in a safe and non-invasive fashion.

Analgesia, Epidural↗
PubMed1997

Release of atrial natriuretic peptide during pulmonary artery clamping in man.

The vasodilating hormone atrial natriuretic peptide (ANP) is secreted from the heart in response to atrial wall stretch, but knowledge of the time course of ANP secretion after acute releasing stimuli is limited. The time from stimulus to release of immunoreactive atrial natriuretic peptide (irANP) was investigated by unilaterally clamping the pulmonary artery in 12 patients undergoing pulmonary surgery. The second messenger to ANP-induced vascular relaxation, plasma cyclic guanosine monophosphate (p-cGMP), was measured as an indirect marker of the vascular effects of ANP. Immediately after applied clamping, p-irANP (baseline level 15.4 +/- 2.9 pmol l-1) started to increase, reaching a significant, although moderate, increase (11 +/- 4%, P < 0.05) after 2 min. This elevation of p-irANP remained during the entire clamping period (+13 +/- 6%, P < 0.05 vs. baseline). Within 1 min of declamping, p-irANP returned to the baseline level. No conclusive alterations in p-cGMP were observed. The prompt ANP response to the applied stimulus, and the return to baseline after declamping, may indicate the presence of a short time-acting releasing mechanism of ANP.

Aged↗
PubMed2002

Replication fork reversal in DNA polymerase III mutants of Escherichia coli: a role for the beta clamp.

Certain replication mutations lead in Escherichia coli to a specific reaction named replication fork reversal: at blocked forks, annealing of the nascent strands and pairing of the template strands form a four-way junction. RuvABC-catalysed resolution of this Holliday junction causes chromosome double-strand breaks (DSBs) in a recBC context and therefore creates a requirement for the recombination proteins RecBC for viability. In the present work, two mutants were tested for replication fork reversal: a dnaEts mutant and a dnaNts mutant, affected in the alpha (polymerase) and beta (processivity clamp) subunits of DNA polymerase III holoenzyme respectively. In the dnaEts recB strain, RuvABC-dependent DSBs caused by the dnaEts mutation occurred at 37 degrees C or 42 degrees C, indicating the occurrence of replication fork reversal upon partial or complete inactivation of the DNA polymerase alpha subunit. DSB formation was independent of RecA, RecQ and the helicase function of PriA. In the dnaNts recB mutant, RuvABC-dependent DSB caused by the dnaNts mutation occurred only at semi-permissive temperature, 37 degrees C, indicating the occurrence of replication fork reversal in conditions in which the remaining activity of the beta clamp is sufficient for viability. In contrast, the dnaNts mutation did not cause chromosome breakage at 42 degrees C, a temperature at which DnaN is totally inactive and the dnaNts mutant is inviable. We propose that a residual activity of the DNA polymerase III beta clamp is required for replication fork reversal in the dnaNts mutant.

Bacterial Proteins↗
PubMed2002

C1-inhibitor reduces the ischaemia-reperfusion injury of skeletal muscles in mice after aortic cross-clamping.

BACKGROUND: Both C1-inhibitor (C1-INH) and antibodies against the CD18 adhesion molecule have been shown to reduce ischaemia-reperfusion injuries. The objective of this study was to investigate the effect of increased ischaemia times and to determine whether inhibiting C1 or blocking the CD18 function was protective in skeletal muscle ischaemia-reperfusion injury after aortic cross-clamping. MATERIALS AND METHODS: BALB/c mice were subjected to aortic cross-clamping below the renal artery for 60, 75 or 105 min, followed by 3 h of reperfusion. Two-thirds of a total dose of anti-CD18 antibody (40 mg/kg) or human C1-INH (1,000 IU/kg) was given by intraperitoneal injection before ischaemia and one-third immediately after the clamping. Creatine kinase (CK) in the plasma was used as an indicator of muscle injury severity. RESULTS: There was a consistent rise in the plasma CK concentration proportional to the length of ischaemia (P < 0.0005). C1-INH treatment significantly (P = 0.012) reduced the plasma CK for the ischaemia times of 75 and 105 min. The anti-CD18 antibody did not have any effect, as demonstrated by the CK values that were similar to controls (P = 0.836). CONCLUSION: The data support a beneficial role for C1-INH in the treatment of ischaemia-reperfusion injuries of skeletal muscles.

Animals↗
PubMed2001

Early clamping of umbilical cord blood and foetal CD34 enrichment.

We describe the relation between clamping time and blood volume collected, and two enrichment systems of CD34+ stem cells from umbilical cord blood, in order to determine the criteria for an excellent recovery with high proliferative ability and bone marrow reconstitution. After an obstetrician-based cord blood collection, the purification of stem cells was performed either with a combination of monoclonal antibodies, negative selections, using the Stem Sep method, or with a positive cells selection owing to their surface CD34 antigens, using the Mini Macs system. An excellent recovery of haematopoietic progenitors--burst forming unit erythroid; colony-forming unit granulocyte and macrophage; and colony-forming unit granulocyte, erythroid, monocyte, and macrophage--inversely related to an increase in clamping time, was achieved using the Mini Macs system (54% of colonies with a 90% purity), while the recovery of haematopoietic progenitors was 35% (with a 80% purity) using the Stem Sep method. By clamping umbilical cord blood at an early stage, we obtained a greater number of CD34+ cells, and their clonogenic activity increased with enrichment. This is particularly useful considering that the number of CD34+ stem cells contained in a unit of placental blood is sufficient for transplanting into a child but not for an adult engraftment. Thus, using these methods, we obtain a larger number of CD34+ stem cells. This increases the possibility of reducing graft versus host disease in adult patients and produces survival rates similar to the ones we observed in transplantation of bone marrow from unrelated donors.

Antigens, CD↗
PubMed2000

Prevention of renal impairment following aortic cross-clamping by manipulation of the endogenous renal nitric oxide response.

OBJECTIVE: Infrarenal aortic cross-clamp-induced lower torso ischaemia-reperfusion injury is associated with impairment of glomerular filtration rate and upregulation of endogenous renal nitric oxide production. The aim of this study was to investigate whether manipulation of the endogenous renal nitric oxide response can ameliorate subsequent renal injury. METHODS: Groups of male Wistar rats (n = 6) were treated with one of the following agents before being subjected to 60 min of infrarenal aortic cross-clamping: saline (control), L-NMMA (a pan nitric oxide synthase inhibitor), 1400W (a highly selective iNOS inhibitor), hydrocortisone (an inhibitor of the systemic inflammatory response), L-arginine (the substrate for nitric oxide synthase) and NOC-18 (a nitric oxide donor). Animals were recovered after a left nephrectomy. The glomerular filtration rate (GFR) of the remaining kidney was measured on the second and seventh postoperative day using a 99Tc DPTA clearance technique as an index of renal injury. RESULTS: Animals treated with L-NMMA prior to aortic cross-clamping had a significantly impaired GFR compared to controls on the second (p<0. 01, Mann-Whitney U -test) and seventh (p<0.05, Mann-Whitney U -test) postoperative day. Hydrocortisone and 1400W had no significant effect on GFR on the second or seventh postoperative day. L-arginine and NOC-18 had no influence on GFR on the second postoperative day but significantly improved GFR on the seventh postoperative day. CONCLUSIONS: These results show that the endogenous nitric oxide response protects the kidneys from ischaemia-reperfusion injury. Manipulation of the renal nitric oxide response may have therapeutic benefits to patients undergoing aortic aneurysm repair by preventing acute renal failure.

Animals↗
PubMed2000

Relationship between intrathecal oxygen tension and ultrastructural changes in the spinal cord during experimental aortic clamping.

OBJECTIVES: To investigate spinal cord ultrastructure related to cerebrospinal fluid (CSF) oxygenation. DESIGN: experimental aortic occlusion model with intrathecal oxygen tension monitoring. MATERIALS AND METHODS: Two groups of pigs underwent proximal (P) or double (D) aortic occlusion for 30 min followed by 1 h of reperfusion. In a third group (I) segmental arteries distal to T3 were clamped for 90 min. A thin pO(2), pCO(2) and pH sensor was placed intrathecally for continuous monitoring of CSF. Spinal cord segments were studied by electron microscopy (EM). RESULTS: In group P, CSF-pO(2)rapidly decreased during clamping and major changes in pH and pCO(2)were seen. EM demonstrated neuronal degeneration with loss of cellular integrity and severe affection of organelles. In the group D, CSF oxygenation decreased to about half, but with only moderate changes in the metabolic parameters. Group I showed no significant changes in CSF measurements. The latter groups were similar at EM, showing only mild mitochondrial changes. CONCLUSIONS: The level of CSF oxygenation during aortic cross-clamping or segmental artery interruption seems to correlate with ultrastructural changes in the spinal cord. This online intrathecal monitoring technique may provide valuable information on spinal cord circulation during thoracoabdominal aortic surgery.

Animals↗
PubMed2003

A porcine model of systemic and renal haemodynamic responses to infrarenal aortic cross-clamping.

OBJECTIVES: cross-clamping of the infrarenal aorta is associated with complex haemodynamic disturbances. Several experimental models of aortic cross-clamping (AXC) have been described with heterogeneous results. The main purpose of this study was to establish an animal model in which infrarenal AXC could reproduce similar systemic and renal haemodynamic changes to those observed in humans. METHODS: eleven anaesthetised pigs underwent AXC just below the renal arteries. Renal blood flow was measured using clearance of (131)I hippuran. Systemic and renal parameters were collected at 3 consecutive 30-min periods. RESULTS: AXC did not alter the extraction fraction of (131)I hippuran but was accompanied by significant (13%) decrease in cardiac index (p = 0.005) and a 23% increase in mean arterial pressure (p = 0.005). AXC induced significant 135% increase in renal vascular resistance (p = 0.012) and a 35% decrease in renal blood flow (p = 0.016). This worsened after removal of the aortic clamp, whereas systemic variables returned to baseline levels. CONCLUSIONS: this AXC animal model reproduces the changes observed in humans. It provides a reliable animal model which allows to investigate the underlying mechanisms of renal vasoconstriction and the effect of new drugs.

Animals↗
PubMed2003

Quantitative analysis of left ventricular regional wall motion with color kinesis during abdominal aortic cross-clamping.

OBJECTIVES: The authors aimed to establish a technique for quantitative analysis of regional wall motion abnormality (RWMA) using color kinesis (CK) of transesophageal echocardiography (TEE) in surgical patients. This technique was used to determine whether RWMAs develop de novo after infrarenal aortic cross-clamping in patients undergoing vascular surgery with a preoperative dipyridamole thallium stress test (DTST). DESIGN: An observational study. SETTING: University hospital. PARTICIPANTS: Thirty-eight patients undergoing infrarenal abdominal aortic aneurysm resection or aortofemoral bypass. MEASUREMENTS AND MAIN RESULTS: CK images of the left ventricle (LV) were obtained from the midventricular transgastric short-axis view before and after infrarenal aortic cross-clamping using TEE and analyzed off-line using custom software. The predictive value of the category "reversible perfusion defect" (RD) was also estimated from DTST for predicting new RWMAs with CK. CK analysis is suitable for clinical use based on the comparison with conventional two-dimensional echocardiogram measurements and interobserver variability. CK analysis showed all 7 patients with persistent perfusion defects on DTST had RWMAs. New RWMAs occurred in 2 of 9 patients with RD and in 2 of 15 patients with normal DTST, indicating that there was no significant difference between RD and normal DTST in the incidence of new RWMAs. CONCLUSIONS: A new method is available for clinical use, which is capable of visualizing RWMAs. These results suggest that new RWMAs introduced by aortic cross-clamping occur irrespective of the risk as assessed by preoperative DTST. CK with the new analysis method might be a useful tool to quantitatively evaluate RWMAs during surgery.

Aorta, Abdominal↗
PubMed1977

Explusion of SALIBI-clamp from the internal carotied artery in the neck by infection.

A left infraclinoid carotid aneurysm (3 x 2-5 x 2 cm) in a 60-year old woman presented with total ophthalmoplegia and trigeminal symptoms. A staged internal carotied occlusion in the neck was performed using a SALIBI-clamp. A chronic infectious fistula developed (Klebsiella aerogenes). When superficial debridement failed, operative removal of the SALIBI-clamp was planned seven months after its insertion. The clamp was found subcutaneously without any relation to the internal carotid artery and was easily removed. Subsequent catheter angiography proved the left internal carotied to be completely occluded in the neck. No cross filling of the aneurysm could be demonstrated from the right carotied nor the vertebro-basilar arteries. The neurological deficit remained unchanged. The wound closed promptly without further signs of infection.

Angiography↗