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At least 721 records · Page 40Linked to original sources
PubMed1999

Comparison of the assembly of the bacteriophage T4 clamp loader complex (gp44/62) expressed in a cis versus trans genomic configuration.

Proper formation of the bacteriophage T4 DNA polymerase holoenzyme requires a wide spectrum of protein-protein and protein-DNA interactions among the DNA polymerase gp43, the sliding clamp gp45, and gp44/62, the clamp loader complex (CLC). The 44 and 62 proteins associate to form a tight complex maintained in a 4:1 ratio. The 44 and 62 genes are adjacent to each other on the T4 genome, are cotranscribed, and are translationally coupled. It has been suggested that translational coupling may play a role in the formation of the clamp loader complex and may control its stoichiometry. To examine the effect of coupling on the assembly of the complex, expression in trans of genes 44 and 62 was accomplished by cotransforming Escherichia coli with compatible, inducible plasmid vectors. A gp44/62 complex could be purified from such cells. The complex assembled in trans exhibited stoichiometry and ATPase activity identical to native complex. Burst sizes were determined to gauge the efficiency of clamp loader complex formation. When gp44 was supplied by a plasmid and gp62 was supplied by the T4 genome, complex formation was as efficient as in wild-type virus. However, when gp62 was supplied by plasmid and gp44 was supplied by the T4 genome, efficiency of complex formation was decreased. This decrease in the efficiency of complex formation was temperature dependent, being more pronounced at higher temperatures. At higher temperatures, a larger proportion of gp62 expressed from the plasmid was found to be present in an insoluble form. The decrease in efficiency of complex formation correlated to a decrease in solubility of the gene 62 protein.

Bacteriophage T4↗
PubMed1992

Patch-clamp recordings of spiking and nonspiking interneurons from rabbit olfactory bulb slices: membrane properties and ionic currents.

Physiological and morphological properties of rabbit, Oryctolagus cuniculus, olfactory bulb interneurons were characterized by using a thin slice preparation in combination with patch-clamp measurements and Lucifer Yellow fills. Two types of interneurons, periglomerular (PG) and juxtaglomerular (JG) cells, were unequivocally distinguished in the glomerular layer. Their properties were compared to those of mitral cells. PG cells closely resembled previously described periglomerular cells in their morphology. During current clamp recording these neurons were characterized by their lack of action potentials upon depolarization. Consistent with these results no Na+ currents could be elicited in voltage clamp experiments. Two types of outward K+ currents were distinguished: one which inactivated and one which did not. From their morphology JG cells appear to be either short axon cells or external tufted cells. JG cells always responded with a single, TTX-blockable action potential in response to maintained current injection. Two types of membrane currents were identified in JG cells during voltage clamp: a fast, inactivating Na+ current that was fully activated at -80 mV, and a sustained outward current that shared some properties with a delayed rectifier K+ current. The particular relationship between the voltage dependence of the Na+ and K+ currents appeared to preclude repetitive spike activity.

Action Potentials↗
PubMedDate not supplied

Voltage clamping with single microelectrodes: comparison of the discontinuous mode and continuous mode using the Axoclamp 2A amplifier.

The voltage clamp technique is a powerful method for studying the physiology of excitable membrane. This technique has made possible the determination of ionic responses generated by activation of either receptor-mediated or voltage-dependent processes. The development of the whole-cell, 'tight-seal' voltage clamp method has allowed the analysis and examination of membrane physiology at the single cell level. The method allows the characterization of voltage-dependent ionic conductances both at the macroscopic (whole-cell) and at the microscopic (unitary conductance or single channel) level in cells less than 10 micron in diameter, a feat difficult to achieve with 'conventional' fine-tipped micropipettes. In this paper, several methologies used for culturing neuronal and non-neuronal cells in the laboratory are described. A comparison between the two modes of voltage clamp using blunt-tipped 'patch'-microelectrodes, the switching (discontinuous) and the non-switching (continuous) modes, of the Axoclamp-2A amplifier is made. Some results on membrane currents obtained from neuronal and non-neuronal cells using the single electrode whole-cell 'tight-seal' voltage clamp is illustrated. The possible existence of two inactivating K+ currents, one dependent on Ca++ the other is not, is discussed.

Amplifiers, Electronic↗
PubMed1987

Reduction of platelet aggregation induced by euglycaemic insulin clamp.

To examine the effect of serum insulin independent of the level of blood glucose in vivo on platelet aggregation in healthy individuals, a euglycaemic insulin clamp was applied up to 4 h. During the clamp, blood glucose at 5.0 mmol/l and insulin levels at 100 microU/ml were maintained. Blood samples were drawn before, 2 and 4 h after the start of the insulin clamp. The platelet aggregation induced by 1 mumol/l and 2 mumol/l ADP, 1 microgram/ml collagen and 2.7 mumol/l epinephrine was measured in the blood samples. Platelet aggregation induced by adenosine diphosphate, collagen and epinephrine in the 4 h sample was significantly reduced from the pre-clamp value of 8.4% to 3.9% (p less than 0.05), 26.2% to 7.0% (p less than 0.01) and 31.8% to 9.1% (p less than 0.01), respectively. On the other hand, when the same individuals were infused with physiological saline and blood glucose (4.4 mmol/l) and insulin level (10 mIU/l) were kept within normal values, there was no difference between the values of induced platelet aggregation in samples drawn before and during the insulin infusion. It was concluded that hyperinsulinaemia reduces platelet aggregation in vivo when euglycaemia was maintained.

Adult↗
PubMed1989

A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system.

(1) A preparation is described which allows patch clamp recordings to be made on mammalian central nervous system (CNS) neurones in situ. (2) A vibrating tissue slicer was used to cut thin slices in which individual neurones could be identified visually. Localized cleaning of cell somata with physiological saline freed the cell membrane, allowing the formation of a high resistance seal between the membrane and the patch pipette. (3) The various configurations of the patch clamp technique were used to demonstrate recording of membrane potential, whole cell currents and single channel currents from neurones and isolated patches. (4) The patch clamp technique was used to record from neurones filled with fluorescent dyes. Staining was achieved by filling cells during recording or by previous retrograde labelling. (5) Thin slice cleaning and patch clamp techniques were shown to be applicable to the spinal cord and almost any brain region and to various species. These techniques are also applicable to animals of a wide variety of postnatal ages, from newborn to adult.

Animals↗
PubMed1988

Ca2+ not cyclic AMP mediates the fluid secretory response to isoproterenol in the rat mandibular salivary gland: whole-cell patch-clamp studies.

We have performed whole-cell patch-clamp studies on dispersed secretory cells of the rat mandibular gland to determine how beta-adrenergic stimulation causes fluid secretion. When the pipette contained a high K+ solution, the resting membrane potential averaged -33 mV +/- 1.1 (SEM, n = 34) and the clamped cell showed strong outward rectification. We monitored K+ and Cl- currents for periods of 15 min by recording the currents needed to clamp the cell potential at 0 and -80 mV, respectively. Isoproterenol (1-2 mumol/l) caused increases in the clamp current at 0 mV (the K+ current) and at -80 mV (the Cl- current) in about 80% of cases, although the responses were variable in size and time-course; the responses were indistinguishable from those induced by acetylcholine or the Ca2+ ionophore, A23187. The alpha-adrenergic antagonist, phentolamine (1-2 mumol/l), had no effect on the response, but the beta-adrenergic antagonist, propranolol (10 mumol/l), blocked it completely. The isoproterenol response could not be mimicked by application to either surface of the cell membrane, of cyclic AMP (100 mumol/l), forskolin (1 or 20 mumol/l) or cholera toxin (2.5 micrograms/ml). However, increasing the Ca2+-chelating capacity of the pipette solution by raising its EGTA concentration from the customary 0.5 to 20 mmol/l, blocked the response to isoproterenol, suggesting that beta-adrenergic agonists activate Cl- and K+ channels by raising cytosolic Ca2+. Since neomycin, which blocks phospholipase C, blocked the action of isoproterenol without impairing the cell responsiveness to A23187, it appears that isoproterenol, like muscarinic agonists, increased cytosolic Ca2+ via the phosphatidylinositol cycle.

Acetylcholine↗
PubMed1988

The use of cytodex microcarrier beads in patch-clamp studies on cultured epithelial cells.

This paper describes an economical attachment substratum for use in patch-clamp experiments on cultured epithelial cells. It consists of Cytodex microcarrier beads on which growing cells present a unique profile perspective that increases the ease with which patch-clamp seals can be obtained. Cells from the mouse mandibular gland cell line, ST885, were grown on the beads and studied using standard patch-clamp techniques. The results were indistinguishable from those obtained from cells grown in monolayers on Petri dishes. The method can be applied in many types of patch-clamp experiment and used with a wide variety of cell types. It is especially useful if one wishes to do experiments in the cell-attached or whole-cell configurations and needs to add drugs or hormones to the external surface of the cell.

Animals↗
PubMed1977

Double sucrose gap voltage clamp in cardiac muscle. Indirect assessment of voltage control from tension records.

In double sucrose gap voltage clamp experimetns on frog atrial bundles the configuration of membrane current and contraction was used to estimate the quality of voltage control. Attention was focused on possible action potential activity along the test segment in response to depolarizing clamps. At low depolarizations large Na+ inward currents were observed while any tension response was missing. Transmembrane potential threshold for generation of a mechanical response was evaluated from conditioned (attenuated) action potentials. The mechanical threshold determined from action potential measurements was only slightly higher than that determined from step-clamp depolarizations. With clamp potentials below the threshold, then, any action potential activity induced by the inward current phase is expected to be rudimentary.

Animals↗
PubMedDate not supplied

Excitation-contraction coupling in voltage-clamped cardiac myocytes.

Myocytes isolated from guinea pig ventricles were voltage-clamped using patch pipettes in the whole-cell configuration. For proper voltage control fast Na+ current was blocked by TTX or inactivated by an appropriate prepulse. Zero-load cell shortening was monitored by a photoelectric device. The mechanical response to a short depolarizing clamp was mainly a phasic (transient) contraction. Long-lasting depolarizations caused a tonic (sustained) shortening of a cell. Different clamp patterns were used to study the mode of activation of phasic contraction. 1) With a constant Ca2+ preload established by a train of conditioning pulses, the shortening-voltage relation measured with test pulses of varying height was a bell-shaped curve reflecting the slow inward current (ICa)-voltage relation. The test pulse had a striking influence on the first contraction of the following conditioning series, resulting in an S-shaped relation between post-test contraction and test potential. 2) With series of identical clamps of varying height, steady-state contraction was maximal around 40 mV and not in proportion to ICa. In these measurements Ca2+ preload was likely to increase with increasing potential. It is concluded that ICa initiates phasic contraction by inducing a release of Ca2+ from internal stores while replenishment of the stores is largely determined by an electrogenic transsarcolemmal Na+-Ca2+ exchange. The data suggest that Na+-Ca2+ exchange is not only involved in long-term changes of cardiac contractility but also in beat-to-beat regulation.

Action Potentials↗
PubMed1992

Management of juxtarenal aortic occlusions: technique for suprarenal clamp placement.

Atherosclerotic occlusion of the entire infrarenal abdominal aorta can produce gangrene, rest pain or claudication and can progress to involve the renal artery origins. Features of the operative technique for treating these juxtarenal aortic occlusions include self-retaining retraction, mobilization of the left renal vein with division of all non-renal branches, exposure of the suprarenal aorta and renal arteries by division between clamps of the surrounding paraaortic fibroareolar tissue and fat, sharp division of crural attachments to the aorta, control of the two renal arteries with doubled vessel loops and then direct vertical clamping of the suprarenal aorta. Through an arteriotomy below the renal arteries, 2-4 cm of pararenal aorta are cleared of thrombus and atherosclerotic debris under direct vision. After transfer of the suprarenal clamp to an infrarenal position, conventional aortobifemoral bypass is then performed. In a series of 18 patients with juxtarenal aortic occlusion managed by this technique, suprarenal clamp time ranged from 4 to 25 minutes (mean, 13 minutes). There was no morbidity from renal failure or emboli and no mortality. This technique allows for deliberate, careful disobliteration of the pararenal and infrarenal aorta and minimizes the risk of renal embolization.

Aorta, Abdominal↗
PubMed1976

Effect of clamping of the renal vein in dogs on certain biochemical and histopathological changes.

Experimental investigations were carried out on dogs in which the renal vein was clamped for a period of 15 min, 30, and 60 min consecutively. It has been found that clamping of the renal vein for 15 min results in tissue anoxia and degeneration of the epithelium of the renal tubules. Clamping for 60 min causes cellular acidosis and necrosis of the tubules. It can therefore be said that clamping of the renal vein for even a short time is highly dangerous.

Animals↗
PubMed1997

Direct measurement of clamping forces in cardiovascular surgery.

Equipment for measuring the direct tip force of a clamp arm on the vessel wall is designed as an adjustable surgical clamp with strain gauges applied and connected via an amplifier to a computer-based data acquisition system. A mechanical model that incorporates the resistance of tissue against momentary deformations is developed to analyse and justify measured results. In in vivo experiments on rat thoracic aorta, the minimum occlusion force, stationary clamping force and the momentary peak clamping force are measured and observed as an important cause of damages in the endothelial layer of vascular walls.

Anastomosis, Surgical↗
PubMed1998

Cross-clamping of the three hepatic veins in the piggyback technique is a safe and well tolerated procedure.

A common stump of the three hepatic veins has always been used to fashion the upper vena cava anastomosis in 205 liver transplantations with the piggyback (PGB) technique performed in our Unit, to avoid outflow problems. The aim was to study the repercussion of lateral inferior vena cava (IVC) clamping on IVC flow and pressure as well as on systemic hemodynamics. We have studied 42 orthotopic liver transplantations performed with the PGB technique. Intraoperative IVC blood flow measurements by transit time ultrasonic volume flowmetry, IVC pressure, and systemic hemodynamics were taken before and after lateral IVC clamping. Graft outflow complications, stenosis or kinking of the upper vena cava anastomosis have not been found in any of the 205 PGB procedures. A significant decrease of IVC flow (23%) and cardiac output (12%) occurred after IVC clamping, whereas mean arterial and central venous pressures were not altered significantly, probably due to an increase (25%) of systemic vascular resistance. Only in one case was an almost total clamping of IVC needed. Venovenous bypass was not needed in any case. Renal perfusion pressure was adequate in all cases. We conclude that the use of a common stump of the three hepatic veins for upper vena cava anastomosis in the PGB technique is safe because any outflow problem of the graft is avoided and, at the same time, is well tolerated hemodynamically because most of the IVC flow is preserved.

Constriction↗
PubMed2006

Anatomical basis for clamping of the right hepatic vein outside the liver during right hepatectomy.

The possibility and value of clamping the right hepatic vein (HV) outside the liver during right hepatectomy remain a matter of debate. We carried out an anatomical study on ten fresh cadaveric subjects with no abdominal scarring or hepatic lesions, to determine the biometry of the extraparenchymatous segment of the right HV. One or several accessory right HVs were found in 90% of cases on release of the right edge of the inferior vena cava (IVC). These accessory right HVs had a diameter greater than that of the superior right HV in 10% of cases. In 70% of cases, the extraparenchymatous segment of the vein was free of collateral branches, and in 30% of cases, it was joined by a branch close to its point of exit from the hepatic parenchyma. The length of the vein that can be clamped (length between the point of exit from the hepatic parenchyma and the point of entry of the right HV into the IVC) was 8.6 +/- 1.8 mm (6-12). The right HV entered the vena cava, at an acute angle, in 100% of cases. Clamping of the right HV was possible in all cases. Knowledge of these anatomical points makes it possible to isolate an extraparenchymatous segment of the right HV more safely. The right HV can be isolated and clamped outside the liver in more than 80% of cases, making it possible to carry out right hepatectomy on an exsanguinous liver.

Aged↗
PubMed2005

Comparison between two methods of drain clamping after total knee arthroplasty.

INTRODUCTION: To study the clinical outcome of two methods of drain clamping after total knee arthroplasty and to determine the effect on blood loss and blood transfusion. PATIENTS AND METHODS: A prospective study involving 73 patients, randomized into two groups. Group 1 included patients in whom the drain was clamped for 1 h postoperatively, after which it was released and kept open for 48 h. Group 2 included patients in whom the drain was clamped and released for 10 min every 2 h for 24 h. The drain was removed at 48 h in both groups. Patient's demographic details, intraoperative and postoperative blood loss, and haemoglobin values on the preoperative and postoperative days (1, 4, 7, 14) were recorded. RESULTS: The mean postoperative drainage in group 1 was 520.65+/-170 ml, which was significantly higher than that of group 2, 367.62+/-141.1 ml (p < 0.05, Student's t-test). The drain charting shows 65% of drainage volume occurs in the first 8 h in both groups. The study suggests a reduction in the incidence of blood transfusion in the 2-hourly groups, although it was not statistically significant. There was a difference in the haemoglobin drop between both groups, but statistically the p value was marginally above 0.05 (p = 0.086) and hence not significant. CONCLUSION: The method of 2-hourly clamping of drain and release for 10 min significantly reduces postoperative blood loss, without any added increase of complication, after total knee arthroplasty.

Aged↗
PubMed2005

Control of severe hemorrhage using C-clamp and arterial embolization in hemodynamically unstable patients with pelvic ring disruption.

INTRODUCTION: Hemorrhage is the leading cause of death in patients with a pelvic fracture. The majority of blood loss derives from injured retroperitoneal veins and broad cancellous bone surfaces. The emergency management of multiply injured patients with pelvic ring disruption and severe hemorrhage remains controversial. Although it is well accepted that the displaced pelvic ring injury must be rapidly reduced and stabilized, the methods by which control of hemorrhagic shock is achieved remain under discussion. It has been proposed to exclusively use external pelvic ring stabilization for control of hemorrhage by producing a 'tamponade effect' of the pelvis. However, the frequency of clinically important arterial bleeding after external fixation of the pelvic ring remains unclear. We therefore undertook this retrospective review to attempt to answer this one important question: How frequently is arterial embolization necessary to control hemorrhage and restore hemodynamic stability after external pelvic ring fixation? MATERIALS AND METHODS: We performed a retrospective review of 55 consecutive patients who presented with unstable types B and C pelvic ring fractures. Those patients designated as being in hemorrhagic shock (defined as a systolic blood pressure less than 90 mmHg after receiving 2 L of intravenous crystalloid) were treated by application of the pelvic C-clamp. Patients who remained in hemorrhagic shock, or were determined to be in severe shock (defined as mandatory catecholamines or more than 12 blood transfusions over 2 h), underwent therapeutic angiography within 24 h in order to control bleeding. RESULTS: Fourteen patients were identified as being hemodynamically unstable (ISS 30.1 +/- 11.3 points) and were treated with a C-clamp. In those patients with persistent hemodynamic instability, arterial embolization was performed. After C-clamp application, 5 of 14 patients required therapeutic angiography to control bleeding. Two patients died, one from multiple sources of bleeding and the other from an open pelvic fracture (total mortality 2/14, 14%). CONCLUSIONS: Although the C-clamp is effective in controlling hemorrhage, one must be aware of the need for arterial embolization to restore hemodynamic stability in a select subgroup of patients.

Adolescent↗
PubMed2006

Sympathectomy for hyperhidrosis: should we place the clamps at T2-T3 or T3-T4?

Endoscopic thoracic sympathectomy is routinely used to treat severe hyperhidrosis. It is usually performed at the T2-T3 level of the nerve, but may produce less severe compensatory hidrosis if performed at a lower level. This study evaluates the outcome of 1,274 patients who underwent endoscopic thoracic sympathectomy for plamar, plantar, axillary or facial hyperhidrosis/blushing. Half of the patients were clamped at the T2-T3 level and half were clamped at the T3-T4 level. Postsurgical symptoms and side effects were assessed by interview. All of patients with palmar hyperhidrosis were cured or improved. Patients with plantar and axillary hyperhidrosis were more likely to be improved at T3-T4 level clamping. Patients with facial hyperhidrosis were more likely to be cured at T2-T3 level, but did show improvement at the T3-T4 level. Overall satisfaction was higher in the T3-T4 group. Some degree of mild compensatory sweating occurred in all patients. However, severe compensatory sweating was more common in the T2-T3 group. Around 2% of patients requested a reversal of their surgery. Endoscopic thoracic sympathectomy is a safe and effective treatment for hyperhidrosis. Clamping at the T3-T4 level has a more successful outcome. In particular, it appears to reduce the incidence of severe compensatory hidrosis.

Adolescent↗
PubMed2003

Right hepatectomies without vascular clamping: report of 87 cases.

BACKGROUND/PURPOSE: Portal triad clamping and total or intermittent hepatic vascular exclusion are usually used to reduce blood loss during major liver resections. We report, in this retrospective study, the results of right hepatectomy without vascular clamping. METHODS: From January 1986 to July 2001, 87 right hepatectomies, including 14 extended right hepatectomies, were performed without vascular clamping. There was 53 men and 34 women, with a mean age of 60.2 +/- 12.5 years. Indications were 58 metastases, 16 hepatocellular carcinomas, 5 cholangiocarcinomas, 4 adenomas, 3 angiomas, and 1 carcinoid tumor. All the procedures were carried out using an ultrasonic dissector and intraoperative ultrasonography with only vascular control (looping of the hepatic pedicle and supra; and infrahepatic vena cava). RESULTS: There were four postoperative deaths and 23 complications (26%), including hepatocellular failure (6), pulmonary complications (6), transient bile leakage (5), digestive bleeding (2), subphrenic abscess (1), inferior vena cava (IVC) thrombosis (1), disseminated intravascular coagulation (DIC; 1), and evisceration (1). Forty-two patients (48%) had no blood transfusion. The mean blood transfusion requirement was 1.5 +/- 2.7 units. The mean operative length was 280 +/- 60 min and the mean hospital stay was 12.8 +/- 8.1 days. Liver function test results were similar to those in other studies on days 1, 4, and 7 postoperatively, with a return to normal values after 1 week. CONCLUSIONS: In our experience with major liver resections, vascular clamping is not necessary.

Adult↗