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PubMed2006

An isolated Hda-clamp complex is functional in the regulatory inactivation of DnaA and DNA replication.

In Escherichia coli, a complex consisting of Hda and the DNA-loaded clamp-subunit of the DNA polymerase III holoenzyme promotes hydrolysis of DnaA-ATP. The resultant ADP-DnaA is inactive for initiation of chromosomal DNA replication, thereby repressing excessive initiations. As the cellular content of the clamp is 10-100 times higher than that of Hda, most Hda molecules might be complexed with the clamp in vivo. Although Hda predominantly forms irregular aggregates when overexpressed, in the present study we found that co-overexpression of the clamp with Hda enhances Hda solubility dramatically and we efficiently isolated the Hda-clamp complex. A single molecule of the complex appears to consist of two Hda molecules and a single clamp. The complex is competent in DnaA-ATP hydrolysis and DNA replication in the presence of DNA and the clamp deficient subassembly of the DNA polymerase III holoenzyme (pol III*). These findings indicate that the clamp contained in the complex is loaded onto DNA through an interaction with the pol III* and that the Hda activity is preserved in these processes. The complex consisting of Hda and the DNA-unloaded clamp may play a specific role in a process proceeding to the DnaA-ATP hydrolysis in vivo.

Adenosine Triphosphatases↗
PubMed2004

Activation of fibrinolytic pathways is associated with duration of supraceliac aortic cross-clamping.

PURPOSE: The cause of the coagulopathy seen with supraceliac aortic cross-clamping (SC AXC) is unclear. SC AXC for 30 minutes results in both clotting factor consumption and activation of fibrinolytic pathways. This study was undertaken to define the hemostatic alterations that occur with longer intervals of SC AXC. METHODS: Seven pigs underwent SC AXC for 60 minutes. Five pigs that underwent infrarenal aortic cross-clamping (IR AXC) for 60 minutes and 11 pigs that underwent SC AXC for 30 minutes served as controls. No heparin was used. Blood samples were drawn at baseline, 5 minutes before release of the aortic clamp, and 5, 30, and 60 minutes after unclamping. Prothrombin time, partial thromboplastin time, platelet count, and fibrinogen concentration were measured as basic tests of hemostatic function. Thrombin-antithrombin complexes were used to detect the presence of intravascular thrombosis. Fibrinolytic pathway activation was assessed with levels of tissue plasminogen activator antigen and tissue plasminogen activator activity, plasminogen activator inhibitor-1 activity, and alpha2-antiplasmin activity. Statistical analysis was performed with the Student t test and repeated measures of analysis of variance. RESULTS: Prothrombin time, partial thromboplastin time, and platelet count did not differ between groups at any time. Fibrinogen concentration decreased 5 minutes (P =.005) and 30 minutes (P =.006) after unclamping in both SC AXC groups, but did not change in the IR AXC group. Thrombin-antithrombin complexes increased in both SC AXC groups, but were not significantly greater than in the IR AXC group. SC AXC for both 30 and 60 minutes produced a significant increase in tissue plasminogen activator antigen during clamping and 5 minutes after clamping. This increase persisted for 30 and 60 minutes after clamp release in the 60-minute SC AXC group. Tissue plasminogen activator activity, however, increased only in the 60-min SC AXC group during clamping (P =.02), and 5 minutes (P =.05) and 30 minutes (P =.06) after unclamping, compared with both control groups. CONCLUSIONS: Thirty and 60 minutes of SC AXC results in similar degrees of intravascular thrombosis and fibrinogen depletion. Although SC AXC for both 30 and 60 minutes leads to activation of fibrinolytic pathways, only 60 minutes of SC AXC actually induces a fibrinolytic state. Fibrinolysis appears to be an important component of the coagulopathy associated with SC AXC, and is related to the duration of aortic clamping. CLINICAL RELEVANCE: The coagulopathy frequently associated with thoracoabdominal aortic aneurysm repair is thought to revolt visceral ischemia-reperfusion. The nature of this coagulopathy is controversial. The current study demonstrates that the major hemostatic alteration associated with supraceliac aortic cross-clamping is activation of fibrinolytic pathways. The magnitude of this fibrinolytic response is directly related to the duration of supraceliac aortic occlusion. Future efforts to treat this coagulopathy may well include judicious use of autofibrinolytic agents.

Animals↗
PubMed1988

The reason for cardiac output reduction after aortic cross-clamping.

The hypothesis that a decrease in cardiac output during infrarenal aortic cross-clamping is related to a decrease in oxygen consumption in the perfused tissues (cross-clamp-adapted oxygen consumption) rather than to deterioration of myocardial performance has been tested. Twenty-two patients undergoing excision of an aortic abdominal aneurysm were randomly divided into two groups of equal number. During aortic cross-clamping, Group 1 patients received nitroglycerin infusion, 1 to 2 micrograms.kg-1.min-1, whereas Group 2 patients did not receive a nitroglycerin infusion. During aortic cross-clamping, cross-clamp-adapted body oxygen consumption decreased equally in both groups by 40 to 42 percent of baseline values, whereas cardiac output decreased by 17 percent in Group 2 but did not change significantly in Group 1. Mixed venous oxygen content increased significantly after induction of anesthesia and prior to aortic cross-clamping in both groups. During cross-clamping, the values of mixed venous oxygen content remained increased in Group 2 and increased further in Group 1. The data support our hypothesis since a decrease in cardiac output was not associated with an increase in filling pressures during aortic cross-clamping, but was instead associated with an increase in mixed venous oxygen content and a decrease in the arteriovenous oxygen content difference. Nitroglycerin infusion was associated with a further increase in mixed venous oxygen content during aortic cross-clamping and a decrease in the arteriovenous oxygen content difference, without a concomitant increase in oxygen utilization.

Aorta, Abdominal↗
PubMed1994

Systemic hypertension induced by aortic cross-clamping: detrimental effects of direct smooth muscle relaxation compared with ganglionic blockade.

PURPOSE: Infrarenal aortic cross-clamping performed during vascular reconstructive procedures is often accompanied by systemic supraclamp hypertension. Much of the disease and death that attend aortic cross-clamping centers around hypertension. Many different strategies have been developed to attenuate intraoperative hypertension, and a host of pharmacologic agents are regularly used to lessen the heart-related, cerebral, and systemic effects of clamp-induced hypertension. This study was performed to evaluate two such strategies; the intravenous administration of either trimethaphan camsylate or nitroprusside. METHODS: We used a highly controllable and reproducible model of aortic cross-clamping in which we have previously shown the hypertension associated with clamping to be an active process mediated by means of a reflex arc. Ten dogs, five treated with nitroprusside (NP group) and five treated with trimethaphan camsylate (TC group), underwent 90 minutes of aortic cross-clamping. During this 90-minute period each group received 30 minutes of antihypertensive therapy. RESULTS: Control mean arterial pressure +/- SEM was 80 +/- 5 mm Hg for both groups and increased to 140 +/- 5 mm Hg with clamp application. With antihypertensive treatment the elevation in mean arterial pressure produced by cross-clamping was reduced to preclamp levels in the TC group and only partially (52%) in the NP group, despite very high doses of nitroprusside. Cardiac output (CO) increased in the NP group by 115% and decreased by 36% in the TC group. This increase in CO translates into a large (101%) increase in cardiac minute work for the NP group. CONCLUSIONS: The attenuation of clamp-induced hypertension by nitroprusside is associated with a dramatic increase in CO and cardiac work whereas the use of trimethaphan camsylate is not. The use of this ganglionic blocker may be more appropriate in this setting.

Animals↗
PubMedDate not supplied

Umbilical cord clamping: beliefs and practices of American nurse-midwives.

The optimal time for umbilical cord clamping after birth remains a critical unknown fact that has implications for the infant, the mother, and science. A national survey was conducted using a randomized sample (n = 303) of the active membership of the ACNM to determine cord clamping practices and beliefs of American nurse-midwives. The response rate was 56%. The respondents fell into three cord clamping categories: early (EC) or before 1 minute (26%); intermediate (IC) or 1 to 3 minutes (35%); and late (LC) or after pulsations cease (33%). The EC group believes that early clamping facilitates management of the newborn. The IC group believes that a moderate delay of clamping allows for a gradual transition to extrauterine circulation, although many think that the timing of cord clamping is not significant. The LC group have strongly held beliefs that late clamping supports physiologic birth processes. The majority of CNMs (87%) place the baby on the mother's abdomen immediately after birth and 96% avoid clamping a nuchal cord whenever possible. Although Varney's Midwifery was cited most frequently as a reference, 78% of the respondents listed no references reflecting, in part, the absence of evidence-based recommendations for cord clamping practices.

Adult↗
PubMed2005

Mapping the interaction of DNA with the Escherichia coli DNA polymerase clamp loader complex.

Sliding clamps are loaded onto DNA by ATP-dependent clamp loader complexes. A recent crystal structure of a clamp loader-clamp complex suggested an unexpected mechanism for DNA recognition, in which the ATPase subunits of the loader spiral around primed DNA. We report the results of fluorescence-based assays that probe the mechanism of the Escherichia coli clamp loader and show that conserved residues clustered within the inner surface of the modeled clamp loader spiral are critical for DNA recognition, DNA-dependent ATPase activity and clamp release. Duplex DNA with a 5'-overhang single-stranded region (corresponding to correctly primed DNA) stimulates clamp release, as does blunt-ended duplex DNA, whereas duplex DNA with a 3' overhang and single-stranded DNA are ineffective. These results provide evidence for the recognition of DNA within an inner chamber formed by the spiral organization of the ATPase domains of the clamp loader.

Base Sequence↗
PubMed1999

Effect of rate of calcium reduction and a hypocalcemic clamp on parathyroid hormone secretion: a study in dogs.

BACKGROUND: The parathyroid hormone (PTH) calcium curve is used to evaluate parathyroid function in clinical studies. However, unanswered questions remain about whether PTH secretion is affected by the rate of calcium reduction and how the maximal PTH response to hypocalcemia is best determined. We performed studies in normal dogs to determine whether (a) the rate of calcium reduction affected the PTH response to hypocalcemia and (b) the reduction in PTH values during a hypocalcemic clamp from the peak PTH value observed during the nadir of hypocalcemia was due to a depletion of stored PTH. METHODS: Fast (30 min) and slow (120 min) ethylenediamine-tetraacetic acid (EDTA) infusions were used to induce similar reductions in ionized calcium. In the fast EDTA infusion group, serum calcium was maintained at the hypocalcemic 30-minute value for an additional 90 minutes (hypocalcemic clamp). To determine whether the reduction in PTH values during the hypocalcemic clamp represented depletion of PTH stores, three subgroups were studied. Serum calcium was rapidly reduced from established hypocalcemic levels in the fast-infusion group at 30 and 60 minutes (after 30 min of a hypocalcemic clamp) and in the slow-infusion group at 120 minutes. RESULTS: At the end of the fast and slow EDTA infusions, serum ionized calcium values were not different (0.84 +/- 0.02 vs. 0.82 +/- 0.03 mM), but PTH values were greater in the fast-infusion group (246 +/- 19 vs. 194 +/- 13 pg/ml, P < 0.05). During the hypocalcemic clamp, PTH rapidly decreased (P < 0.05) to value of approximately 60% of the peak PTH value obtained at 30 minutes. A rapid reduction in serum calcium from established hypocalcemic levels at 30 minutes did not stimulate PTH further, but also PTH values did not decrease as they did when a hypocalcemic clamp was started at 30 minutes. At 60 minutes, the reduction in serum calcium increased (P < 0.05) PTH to peak values similar to those before the hypocalcemic clamp. The reduction in serum calcium at 120 minutes in the slow EDTA infusion group increased PTH values from 224 +/- 11 to 302 +/- 30 pg/ml (P < 0.05). CONCLUSIONS: These results suggest that (a) the reduction in PTH values during the hypocalcemic clamp may not represent a depletion of PTH stores. (b) The use of PTH values from the hypocalcemic clamp as the maximal PTH may underestimate the maximal secretory capacity of the parathyroid glands and also would change the analysis of the PTH-calcium curve, and (c) the PTH response to similar reductions in serum calcium may be less for slow than fast reductions in serum calcium.

Animals↗
PubMed1999

Should initial clamping for abdominal aortic aneurysm repair be proximal or distal to minimise embolisation?

OBJECTIVES: to determine whether clamping proximally or distally on the infrarenal aorta during abdominal aortic aneurysm (AAA) repair increases the overall embolic potential. MATERIALS AND METHODS: a sheath was placed in the mid-infrarenal aorta of 16 dogs. In eight animals a cross-clamp was placed at the aortic trifurcation, and in another eight animals it was placed in the immediate subrenal position. Under fluoroscopy blood flow within the infrarenal aorta was evaluated by contrast and particle injections. Grey-scale analysis was used to calculate contrast density. Particle distribution was followed fluoroscopically and confirmed pathologically. RESULTS: fifty-seven+/-24% of injected contrast remained within the aorta with distal clamping while 97+/-7% did so with proximal clamping (p<0.01). With distal aortic clamping 6.2+/-1. 3 out of 10 injected particles remained within the aorta after 15 seconds and only 0.8+/-0.8 remained after 5 min. With proximal aortic clamping, all 10 of the particles remained within the aortic lumen for the full 5 minutes (p<0.001). CONCLUSIONS: initial distal clamping minimises distal embolisation, but may result in renal and/or visceral embolisation. Initial proximal clamping prevents proximal embolisation and does not promote distal embolisation. We recommend initial proximal clamping in aortic aneurysm surgery to minimise the overall risk of embolisation.

Animals↗
PubMed2000

DNA structure requirements for the Escherichia coli gamma complex clamp loader and DNA polymerase III holoenzyme.

The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis. Underlying high processivity is a ring-shaped protein, the beta clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template. The beta clamp is assembled onto DNA by the multiprotein gamma complex clamp loader that opens and closes the beta ring around DNA in an ATP-dependent manner. This study examines the DNA structure required for clamp loading action. We found that the gamma complex assembles beta onto supercoiled DNA (replicative form I), but only at very low ionic strength, where regions of unwound DNA may exist in the duplex. Consistent with this, the gamma complex does not assemble beta onto relaxed closed circular DNA even at low ionic strength. Hence, a 3'-end is not required for clamp loading, but a single-stranded DNA (ssDNA)/double-stranded DNA (dsDNA) junction can be utilized as a substrate, a result confirmed using synthetic oligonucleotides that form forked ssDNA/dsDNA junctions on M13 ssDNA. On a flush primed template, the gamma complex exhibits polarity; it acts specifically at the 3'-ssDNA/dsDNA junction to assemble beta onto the DNA. The gamma complex can assemble beta onto a primed site as short as 10 nucleotides, corresponding to the width of the beta ring. However, a protein block placed closer than 14 base pairs (bp) upstream from the primer 3' terminus prevents the clamp loading reaction, indicating that the gamma complex and its associated beta clamp interact with approximately 14-16 bp at a ssDNA/dsDNA junction during the clamp loading operation. A protein block positioned closer than 20-22 bp from the 3' terminus prevents use of the clamp by the polymerase in chain elongation, indicating that the polymerase has an even greater spatial requirement than the gamma complex on the duplex portion of the primed site for function with beta. Interestingly, DNA secondary structure elements placed near the 3' terminus impose similar steric limits on the gamma complex and polymerase action with beta. The possible biological significance of these structural constraints is discussed.

Bacterial Proteins↗
PubMed2003

The transducer domain is important for clamp operation in human DNA topoisomerase IIalpha.

DNA topoisomerase II is a multidomain homodimeric enzyme that changes DNA topology by coupling ATP hydrolysis to the transport of one DNA helix through a transient double-stranded break in another. The process requires dramatic conformational changes including closure of an ATP-operated clamp, which is comprised of two N-terminal domains from each protomer. The most N-terminal domain contains the ATP-binding site and is directly involved in clamp closure, undergoing dimerization upon ATP binding. The second domain, the transducer domain, forms the walls of the N-terminal clamp and connects the clamp to the enzyme core. Although structurally conserved, it is unclear whether the transducer domain is involved in clamp mechanism. We have purified and characterized a human topoisomerase II alpha enzyme with a two-amino acid insertion at position 408 in the transducer domain. The enzyme retains both ATPase and DNA cleavage activities. However, the insertion, which is situated far from the N-terminal dimerization area, severely disrupts the function of the N-terminal clamp. The clamp-deficient enzyme is catalytically inactive and lacks most aspects of interdomain communication. Surprisingly, it seems to have retained the intersubunit communication, allowing it to bind ATP cooperatively in the presence of DNA. The results show that even distal parts of the transducer domain are important for the dynamics of the N-terminal clamp and furthermore indicate that stable clamp closure is not required for cooperative binding of ATP.

Adenosine Triphosphatases↗
PubMed2004

Requirement for ATP by the DNA damage checkpoint clamp loader.

The DNA damage clamp loader replication factor C (RFC-Rad24) consists of the Rad24 protein and the four small Rfc2-5 subunits of RFC. This complex loads the heterotrimeric DNA damage clamp consisting of Rad17, Mec3, and Ddc1 (Rad17/3/1) onto partial duplex DNA in an ATP-dependent manner. Interactions between the clamp loader and the clamp have been proposed to mirror those of the replication clamp loader RFC and the sliding clamp proliferating cell nuclear antigen (PCNA). In that system, three ATP molecules bound to the Rfc2, Rfc3, and Rfc4 subunits are necessary and sufficient for efficient loading of PCNA, whereas ATP binding to Rfc1 is not required. In contrast, in this study, we show that mutant RFC-Rad24 with a rad24-K115E mutation in the ATP-binding domain of Rad24 shows defects in the ATPase of the complex and is defective for interaction with Rad17/3/1 and for loading of the checkpoint clamp. A similar defect was measured with a mutant RFC-Rad24 clamp loader carrying a rfc4K55R ATP-binding mutation, whereas the rfc4K55E clamp loader showed partial loading activity, in agreement with genetic studies of these mutants. These studies show that ATP utilization by the checkpoint clamp/clamp loader system is effectively different from that by the structurally analogous replication system.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Competitive processivity-clamp usage by DNA polymerases during DNA replication and repair.

Protein clamps are ubiquitous and essential components of DNA metabolic machineries, where they serve as mobile platforms that interact with a large variety of proteins. In this report we identify residues that are required for binding of the beta-clamp to DNA polymerase III of Escherichia coli, a polymerase of the Pol C family. We show that the alpha polymerase subunit of DNA polymerase III interacts with the beta-clamp via its extreme seven C-terminal residues, some of which are conserved. Moreover, interaction of Pol III with the clamp takes place at the same site as that of the delta-subunit of the clamp loader, providing the basis for a switch between the clamp loading machinery and the polymerase itself. Escherichia coli DNA polymerases I, II, IV and V (UmuC) interact with beta at the same site. Given the limited amounts of clamps in the cell, these results suggest that clamp binding may be competitive and regulated, and that the different polymerases may use the same clamp sequentially during replication and repair.

Amino Acid Sequence↗
PubMed1993

Spinal cord perfusion pressure in dogs after control of proximal aortic hypertension during thoracic aortic cross-clamping with esmolol or sodium nitroprusside.

BACKGROUND: Spinal cord perfusion pressure may be reduced when sodium nitroprusside is used to control proximal aortic hypertension during thoracic aortic clamping. The effect of esmolol infusion on spinal cord perfusion pressure during thoracic aortic clamping is unknown. This study compares spinal cord perfusion pressure following control of proximal hypertension with either sodium nitroprusside or esmolol during thoracic aortic clamping. METHODS: The thoracic aorta was cross-clamped for 30 min in 18 dogs anesthetized with halothane. A control group (n = 6) received no treatment of proximal hypertension during cross-clamping. In two other groups, proximal arterial pressure was controlled (100 mmHg) by infusion of either sodium nitroprusside (n = 6) or esmolol (n = 6). Brachial and femoral arterial pressures, spinal cord perfusion pressure, pulmonary artery occlusion, central venous pressures, and cardiac output were monitored. Neurologic assessment was performed 24 h following surgery. RESULTS: Femoral arterial pressure was lower with nitroprusside (14 +/- 3 mmHg) compared to esmolol (24 +/- 4 mmHg) after 15 min of aortic cross-clamping. Cerebrospinal fluid pressure increased during aortic cross-clamping in the sodium nitroprusside group (from 7 +/- 5 to 16 +/- 6 mmHg) but not in esmolol or control groups. Spinal cord perfusion pressure was lower with nitroprusside at 15 min of aortic cross-clamping (2 +/- 4 mmHg) compared to control (15 +/- 7 mmHg) and esmolol groups (17 +/- 11 mmHg). Esmolol infusion reduced cardiac output and increased ventricular filling pressures compared to control and nitroprusside groups. CONCLUSIONS: Esmolol was associated with greater spinal cord perfusion pressure, but adverse hemodynamic effects, when compared with nitroprusside during thoracic aortic cross-clamping. When only surviving dogs (4 control, 5 esmolol, 6 nitroprusside) are considered, the incidence of neurologic deficit was greater in nitroprusside-treated dogs than in either control or esmolol-treated dogs. No difference in outcome was present when all dogs are considered.

Adrenergic beta-Antagonists↗
PubMed1992

Force production in voltage-clamped human atrial muscle.

Human atrial muscle preparations obtained during open heart surgery were mounted in a sucrose gap. Force and membrane currents were recorded during voltage clamp. After a 20-s rest, 10 clamps from a holding potential of -40 to 0 mV at 1.0 Hz were given. This was followed by a test clamp (called 1) of a varied duration and amplitude and two more test clamps (called 2 and 3) as during the priming period. Peak force of contraction 1 (F1) was independent of clamp duration from 2s to about 100 ms but declined at shorter durations. Peak force of contraction 2 (F2) and 3 (F3) increased with the duration and became potentiated. Increasing the clamp amplitude raised F1 to an optimum value at about +10 mV and there was a decline at higher voltages. Both F2 and F3 increased at higher amplitudes. A conventional bell-shaped current-voltage relation for the second inward current was obtained during clamp 1 with maximum inward current around -10 mV. In control experiments on isolated human myocytes peak current was recorded at somewhat more positive potentials. The relation between F3 and F2 was linear both when duration and amplitude of clamp 1 was varied. The slope of the line, interpreted as a measure of recirculation of activator calcium, was 0.4. It is concluded that force during voltage clamp in human atrial muscle is similarly related to membrane voltage as previously reported for guinea pig and ferret preparations.

Atrial Function↗
PubMed2002

A three-dimensional finite element model for arterial clamping.

Clamp induced injuries of the arterial wall may determine the outcome of surgical procedures. Thus, it is important to investigate the underlying mechanical effects. We present a three-dimensional finite element model, which allows the study of the mechanical response of an artery-treated as a two-layer tube-during arterial clamping. The important residual stresses, which are associated with the load-free configuration of the artery, are also considered. In particular, the finite element analysis of the deformation process of a clamped artery and the associated stress distribution is presented. Within the clamping area a zone of axial tensile peak-stresses was identified, which (may) cause intimal and medial injury. This is an additional injury mechanism, which clearly differs from the commonly assumed wall damage occurring due to compression between the jaws of the clamp. The proposed numerical model provides essential insights into the mechanics of the clamping procedure and the associated injury mechanisms. It allows detailed parameter studies on a virtual clamped artery, which can not be performed with other methodologies. This approach has the potential to identify the most appropriate clamps for certain types of arteries and to guide optimal clamp design.

Anisotropy↗
PubMed2006

Effect of delayed sampling on umbilical cord arterial and venous lactate and blood gases in clamped and unclamped vessels.

BACKGROUND: Cord blood lactate at birth is a marker of antenatal hypoxia, and is comparable to pH as a prognostic tool. OBJECTIVE: To determine, by a prospective observational study, the effect of delayed sampling from arteries and veins that were double clamped to isolate the blood from the placenta (clamped), and from vessels that were not isolated from the placenta (unclamped). METHODS: Paired samples taken from clamped and unclamped vessels at 0, 20, 40, and 60 minutes were analysed for lactate, base excess, pH, and Pco(2). Data were analysed as the change from time 0 at 20, 40, and 60 minutes. RESULTS: Thirty eight placentas of infants delivered by elective caesarean section were studied. Arterial samples were taken from 20 placentas, and venous samples from 18 placentas. Arterial and venous lactate was significantly higher than at time 0 by 20 minutes in both clamped and unclamped vessels. Changes in unclamped vessels were greater than in clamped vessels. The pH remained unchanged over 60 minutes in clamped vessels, but changed significantly in unclamped vessels. Base excess changed significantly in both clamped and unclamped vessels. CONCLUSIONS: Cord blood samples taken after 20 minutes delay are unreliable for lactate measurement, even if the vessel has been doubly clamped to isolate the blood from the placenta. Current guidelines that state that blood can be sampled from a clamped cord for up to one hour after delivery should not apply to the interpretation of lactate or base excess. Delayed sampling from unclamped cords is very unreliable.

Acid-Base Equilibrium↗
PubMed1993

Analysis of voltage-dependent membrane currents in spatially extended neurons from point-clamp data.

1. Numerical methods were used to evaluate voltage space-clamp performance in the investigation of a voltage-dependent inward current similar to the noninactivating Ca current. In addition, the cell is equipped with a repolarizing system, represented by leak and outwardly rectifying outward conductances. The electrotonically compact model cell is represented by a cable with an electrotonic length of 1 space constant under control conditions, but that becomes effectively only 0.33 space constants during a 90% reduction of the leak and outward conductance. The cable is perfectly voltage clamped at one end. 2. The apparent voltage dependence, activation, and inactivation of the clamp current depend on the distribution of the membrane slope conductance along the cable; this depends on 1) the distribution of the inward current along the cable and 2) the amplitude of the inward current relative to the amplitudes of the leak and voltage-dependent outward currents. 3. Under control conditions, the membrane voltage decays steeply with distance from the command voltage at the clamp site to almost resting potential for most of the rest of the cable. This is because the leak and outward current are dominant over the inward current. The inward current is activated primarily at the clamped part of the cable. Clamp currents are activated instantaneously. The clamp-current current-voltage (I-V) relation is less steep with depolarization because the membrane potential for locations away from the clamp site lags behind the clamp potential. 4. When the conductances for leak and outward current are reduced by 90%, these conductances lose their dominance. The membrane slope conductance now has a range with negative values.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Differential effects of a perioperative hyperinsulinemic normoglycemic clamp on the neurohumoral stress response during coronary artery surgery.

BACKGROUND: Hyperglycemia in patients undergoing coronary artery bypass grafting (CABG) is associated with adverse outcome. Although insulin infusion strategies are increasingly used to improve outcome, a pathophysiological rationale is currently lacking. The present study was designed to quantify the effects of a perioperative hyperinsulinemic normoglycemic clamp on the neurohumoral stress response during CABG. METHODS: Forty-four nondiabetic patients, scheduled for elective CABG, were randomized to either a control group (n = 22) receiving standard care or to a clamp group (n = 22) receiving additionally a perioperative hyperinsulinemic (regular insulin at a fixed rate of 0.1 IU.kg(-1).h(-1)) normoglycemic (plasma glucose between 3.0 and 6.0 mmol.liter(-1)) clamp during 26 h. We measured the endocrine response of the hypothalamus-pituitary-adrenal (HPA) axis, the sympathoadrenal axis, and glucagon, as well as plasma glucose and insulin at regular intervals from the induction of anesthesia at baseline through the end of the second postoperative day (POD). RESULTS: There were no differences in clinical outcome between the groups. In the control group, hyperglycemia developed at the end of surgery and remained present until the final measurement point on POD2, whereas plasma insulin levels remained unchanged until the morning of POD1. In the intervention group, normoglycemia was well maintained during the clamp, whereas insulin levels ranged between 600 and 800 pmol.liter(-1). In both groups, plasma ACTH and cortisol increased from 6 h after discontinuation of cardiopulmonary bypass onward. However, during the clamp period, a marked reduction in the HPA axis response was found in the intervention group, as reflected by a 47% smaller increase in area under the curve in plasma ACTH (P = 0.035) and a 27% smaller increase in plasma cortisol (P = 0.002) compared with the control group. Compared with baseline, epinephrine and norepinephrine increased by the end of the clamp interval until POD2 in both groups. Surprisingly, the area under the curve of epinephrine levels was 47% higher (P = 0.026) after the clamp interval in the intervention group as compared with the control group. CONCLUSION: A hyperinsulinemic normoglycemic clamp during CABG delays and attenuates the HPA axis response during the first 18 h of the myocardial reperfusion period, whereas after the clamp, plasma epinephrine is higher. The impact of delaying cortisol responses on clinical outcome of CABG remains to be elucidated.

Adrenocorticotropic Hormone↗