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At least 595 records · Page 33Linked to original sources
PubMed1992

Atlantoaxial arthrodesis using interlaminar clamps. An improved technique.

The atlantoaxial articulation is a multiaxial joint with a freedom of motion that presents unique problems in achieving surgical stability. Several methods of atlantoaxial arthrodesis have been established with varying degrees of immediate and sustained stability and safety. This study describes a new, improved method of posterior atlantoaxial arthrodesis, using (Halifax) interlaminar clamps with interposed bone graft, which provides distinct benefits over existing techniques. This method achieved atlantoaxial union within 12 weeks in 80% of the study's patients (20/25). The clamp fixation was revised in two patients, with subsequent fusion. Three other patients required revision surgery for nonunion due to loosening of the clamp. This was usually caused by incomplete tightening of the screws, with subsequent loosening or dislocation of the device. Possible clamp loosening is a potential problem with the currently available system, and may be a result of the clamp design. Techniques to minimize this risk and suggestions for improvement in the device are discussed. Nonunion is more likely to develop in patients with rheumatoid arthritis. If nonunion occurs, the pseudarthrosis is generally between the posterior bone graft and the ring of C1.

Atlanto-Axial Joint↗
PubMed2004

Impact of temporary hilar clamping during laparoscopic partial nephrectomy on postoperative renal function: a prospective study.

PURPOSE: Laparoscopic partial nephrectomy for small renal tumors has been increasingly performed in the last few years. We prospectively evaluated preoperative and postoperative differential renal function by renal scan in patients with contralaterally functioning kidneys who underwent laparoscopic partial nephrectomy with hilar clamping. MATERIALS AND METHODS: From July 2002 to June 2003, 17 consecutive patients were included in this prospective protocol and underwent laparoscopic partial nephrectomy for exophytic tumors using en bloc hilar clamping. Preoperative renal scan with differential function was performed 1 month before and 3 months after surgery in all patients. technetium labeled diethylenetetraminepentaacetic acid scan was performed in all patients. RESULTS: Mean warm ischemia time was 22.50 +/- 9.78 minutes (range 10 to 44). Preoperative differential renal function and glomerular filtration rate (GFR) in the affected kidneys were 50.20% +/- 4.90% (range 43 to 58) and 75.56 +/- 16.45 ml per minutes (range 39.4 to 105). At postoperative month 3 differential renal function and GFR in the affected kidney were 48.07% +/- 7.16% (range 39% to 63%) and 72.03 +/- 18.17 ml per minutes (range 31 to 101). There was a nonsignificant negative association between hilar clamp time and change in renal function (postoperative - preoperative) of the affected kidney (r = -0.26, p = 0.31), and a positive correlation between clamp time and change in GFR (r = 0.39, p = 0.12) that did not reach statistical significance. CONCLUSIONS: In patients with contralaterally functioning kidney, temporary hilar clamping with a mean warm ischemia time of 22.5 minutes results in preservation of renal function in the affected kidney. Larger studies with longer followup are necessary to study the impact of warm ischemia further.

Adult↗
PubMed2005

Complete versus selective portal triad clamping for minor liver resections: a prospective randomized trial.

OBJECTIVE: To evaluate the feasibility, safety, efficacy, amount of hemorrhage, postoperative complications, and ischemic injury of selective clamping in patients undergoing minor liver resections. SUMMARY BACKGROUND DATA: Inflow occlusion can reduce blood loss during hepatectomy. However, Pringle maneuver produces ischemic injury to the remaining liver. Selective hemihepatic vascular occlusion technique can reduce the severity of visceral congestion and total liver ischemia. PATIENTS AND METHODS: Eighty patients undergoing minor hepatic resection were randomly assigned to complete clamping (CC) or selective clamping (SC). Hemodynamic parameters, including portal pressure and the hepatic venous pressure gradient (HVPG), were evaluated. The amount of blood loss, measurements of liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), and postoperative evolution were also recorded. RESULTS: No differences were observed in the amount of hemorrhage (671 +/- 533 mL versus 735 +/- 397 mL; P = 0.54) or the patients that required transfusion (10% versus 15%; P = 0.55). There were no differences on postoperative morbidity between groups (38% versus 29%; P = 0.38). Cirrhotic patients with CC had significantly higher ALT (7.7 +/- 4.6 versus 4.5 +/- 2.7 mukat/L, P = 0.01) and AST (10.2 +/- 8.7 versus 4.9 +/- 2.1 mukat/L; P = 0.03) values on the first postoperative day than SC. The multivariate analysis demonstrated that high central venous pressure, HVPG >10 mm Hg, and intraoperative blood loss were independent factors related to morbidity. CONCLUSIONS: Both techniques of clamping are equally effective and feasible for patients with normal liver and undergoing minor hepatectomies. However, in cirrhotic patients selective clamping induces less ischemic injury and should be recommended. Finally, even for minor hepatic resections, central venous pressure, HVPG, and intraoperative blood loss are factors related to morbidity and should be considered.

Aged↗
PubMed2002

Molecular modeling-based analysis of interactions in the RFC-dependent clamp-loading process.

Replication and related processes in eukaryotic cells require replication factor C (RFC) to load a molecular clamp for DNA polymerase in an ATP-driven process, involving multiple molecular interactions. The detailed understanding of this mechanism is hindered by the lack of data regarding structure, mutual arrangement, and dynamics of the players involved. In this study, we analyzed interactions that take place during loading onto DNA of either the PCNA clamp or the Rad9-Rad1-Hus1 checkpoint complex, using computationally derived molecular models. Combining the modeled structures for each RFC subunit with known structural, biochemical, and genetic data, we propose detailed models of how two of the RFC subunits, RFC1 and RFC3, interact with the C-terminal regions of PCNA. RFC1 is predicted to bind PCNA similarly to the p21-PCNA interaction, while the RFC3-PCNA binding is proposed to be similar to the E. coli delta-beta interaction. Additional sequence and structure analysis, supported by experimental data, suggests that RFC5 might be the third clamp loader subunit to bind the equivalent PCNA region. We discuss functional implications stemming from the proposed model of the RFC1-PCNA interaction and compare putative clamp-interacting regions in RFC1 and its paralogs, Rad17 and Ctf18. Based on the individual intermolecular interactions, we propose RFC and PCNA arrangement that places three RFC subunits in association with each of the three C-terminal regions in PCNA. The two other RFC subunits are positioned at the two PCNA interfaces, with the third PCNA interface left unobstructed. In addition, we map interactions at the level of individual subunits between the alternative clamp loader/clamp system, Rad17-RFC(2-5)/Rad9-Rad1-Hus1. The proposed models of interaction between two clamp/clamp loader pairs provide both structural framework for interpretation of existing experimental data and a number of specific findings that can be subjected to direct experimental testing.

Amino Acid Sequence↗
PubMed2005

Glycodelin responses to hyperinsulinaemic clamp vary according to basal serum glycodelin concentration.

OBJECTIVE: Treatment with metformin, an insulin-lowering agent, increases serum glycodelin, a progesterone-regulated lipocalin protein of the reproductive axis that may play a role in foeto-maternal defence mechanisms. This finding led to the hypothesis that insulin might decrease serum glycodelin concentration. DESIGN, PATIENTS AND MEASUREMENTS: Euglycaemic hyperinsulinaemic clamp experiments (n = 50) were carried out on 28 women of reproductive age (range 25-47 years; mean +/- SEM 39 +/- 1.0 years), and the results were analysed with respect to their baseline serum progesterone (< 10 or > or = 10 nmol/l) and glycodelin (< 10 or > or = 10 microg/l, equivalent to < 357 or > or = 357 pmol/l) concentrations at the onset of the clamp. Ten clamp experiments were performed on five women wearing a levonorgestrel-releasing intrauterine device (IUD), and these were analysed as a separate group. RESULTS: Contrary to the hypothesis, no acute glycodelin-lowering effect of insulin was found in any of the groups studied. All the small rises in glycodelin levels detected during acute hyperinsulinaemia occurred in the comparisons of medians and not means, and all such changes took place within the limits seen in the women with no progesterone exposure. In the group with low progesterone/low glycodelin (n = 21), glycodelin showed a small but significant increase at 30 and 90 min of the clamp (P < 0.01). In the group with elevated progesterone/low glycodelin (n = 11), there was a slight glycodelin increase at 30 min (P < 0.05), whereas no increase was found in the group with elevated glycodelin levels (n = 8). In the clamp experiments on women with levonorgestrel-releasing IUD, the basal glycodelin level was low in all cases and, as in the other women with low glycodelin levels, glycodelin was slightly increased at 30, 60 and 90 min of hyperinsulinaemia (P < 0.01). CONCLUSIONS: The results rule out any acute glycodelin-reducing effects of insulin, although indirect long-term effects mediated by insulin on glycodelin secretion cannot be excluded.

Adult↗
PubMed1989

An investigation of possible iatrogenic damage caused by metal rubber dam clamps.

Rubber dam may be held in place over a tooth by metal clamps. It has been shown that a mismatch of contact between the clamp gripping edge and the tooth surface may be reduced to a point contact, thereby concentrating the gripping force generated by the bow of the clamp. Experiments were conducted to measure the force. Clinically realistic loading of the tooth surface by sections of the gripping edge of clamps was carried out using special apparatus. Examination by scanning electron microscopy showed that iatrogenic damage to the tooth could occur. Therefore a plea is made for clamps to be redesigned to reduce any harm.

Dental Instruments↗
PubMed2004

Use of a molar clamp to isolate two adjacent single-rooted teeth: a clinical aid.

AIM: To emphasize the importance of rubber dam isolation in root canal treatment and to present a technique to simultaneously isolate two single-rooted teeth using a molar clamp. SUMMARY: Isolation of two adjacent teeth with inadequate tooth structure is a challenge. The split-dam technique or using two clamps have drawbacks. This report presents a technique to simultaneously isolate two adjacent single-rooted teeth that are difficult to isolate by conventional methods. Its advantages are efficiency, reduction of radiation exposure, better access, good visibility and isolation. KEY LEARNING POINTS: The basis of this technique is to isolate two teeth as two roots of a molar tooth. The indications for this technique are two adjacent prepared crowns and crownless roots. The appropriate molar clamp can be selected on the basis of the mesial-distal clamp jaw distance being equal to the mesial-distal spacing of the two roots. The buccal-lingual jaw distance of the clamp should be smaller than the dimension of the roots to provide adequate retention.

Humans↗
PubMed2001

An in vivo nitric oxide clamp to investigate the influence of nitric oxide on continuous umbilical blood flow during acute hypoxaemia in the sheep fetus.

1. The aims of this study in the ovine fetus were to (1) characterise continuous changes in umbilical blood flow and vascular conductance during acute hypoxaemia and (2) determine the effects of nitric oxide blockade on umbilical blood flow and vascular conductance during normoxic and hypoxaemic conditions using a novel in vivo 'nitric oxide clamp'. 2. Under 1-2% halothane anaesthesia, seven ovine fetuses were instrumented between 118 and 125 days of gestation (term is ca 145 days) with vascular and amniotic catheters and a flow probe around an umbilical artery. At least 5 days after surgery, all fetuses were subjected to a 3 h protocol: 1 h of normoxia, 1 h of hypoxaemia and 1 h of recovery during fetal I.V. infusion with saline or, 1-2 days later, during combined fetal treatment with the nitric oxide (NO) inhibitor N (G)-nitro-L-arginine methyl ester (L-NAME, 100 mg x kg(-1)) and the NO donor sodium nitroprusside (NP, 5.1 +/- 2.0 microg x kg(-1) x min(-1), the 'nitric oxide clamp'). Following the end of the 3 h experimental protocol, the infusion of NP was withdrawn to unmask any persisting effects of fetal treatment with L-NAME alone. 3. During acute hypoxaemia, the reduction in arterial partial pressure of O2 (Pa,O2) was similar in fetuses infused with saline or treated with the nitric oxide clamp. In all fetuses, acute hypoxaemia led to a progressive increase in mean arterial blood pressure and a fall in heart rate. In saline-infused fetuses, acute hypoxaemia led to a rapid, but transient, decrement in umbilical vascular conductance. Thereafter, umbilical vascular conductance was maintained and a significant increase in umbilical blood flow occurred, which remained elevated until the end of the hypoxaemic challenge. In contrast, while the initial decrement in umbilical vascular conductance was prevented in fetuses treated with the nitric oxide clamp, the increase in umbilical blood flow during hypoxaemia was similar to that in fetuses infused with saline. After the 1 h recovery period of the acute hypoxaemia protocol, withdrawal of the sodium nitroprusside infusion from fetuses undergoing the nitric oxide clamp led to a significant, but transient, hypertension and a sustained umbilical vasoconstriction. 4. In conclusion, the data reported in this study of unanaesthetised fetal sheep (1) show that minute-by-minute analyses of haemodynamic changes in the umbilical vascular bed reveal an initial decrease in umbilical vascular conductance at the onset of hypoxaemia followed by a sustained increase in umbilical blood flow for the duration of the hypoxaemic challenge, (2) confirm that the increase in umbilical blood flow after 15 min hypoxaemia is predominantly pressure driven, and (3) demonstrate that nitric oxide plays a major role in the maintenance of umbilical blood flow under basal, but not under acute hypoxaemic, conditions.

Acid-Base Equilibrium↗
PubMed1995

Aortic spoon-jaw clamp for aorto-saphenous vein anastomosis.

In the course of coronary bypass operations the proximal anastomosis of the saphenous vein to the aorta is commonly done either with the aorta clamped completely or by using a partially occluding clamp. While under usual circumstances, either method is feasible; under other conditions, such as in the presence of partial calcification, heavy atherosclerotic deposits, or in reoperations, it is preferable not to perform either of the above methods. The purpose of this article is to present an instrument designed to allow performance of the proximal anastomosis without application of any aortic clamping in the following manner. A relatively healthy spot on the ascending aorta is selected. Under protection of a mattress suture, a stab wound is made on the aortic wall, and the lower jaw of the instrument is inserted through it into the aortic lumen. A spot is selected for the anastomotic site, the instrument is placed to that area, and its handle is closed. This effectively isolates a 10 x 8 mm area of the aortic wall from the blood flow. An opening is made in the area protected by the clamp. With the jaws of the clamp closed the vein-to-aorta anastomosis is completed as usual.

Anastomosis, Surgical↗
PubMedDate not supplied

The micro-mitral operation comparing the Port-Access technique and the transthoracic clamp technique.

BACKGROUND: Several minimally invasive approaches to the mitral valve have been described, including parasternal incision and right anterolateral thoracotomy. MATERIAL AND METHODS: Since September 1996, 58 patients underwent minimally invasive mitral valve surgery at our institution through a right anterolateral minithoractomy. Two different techniques were used for institution of cardiopulmonary bypass (CPB) and aortic clamping: in the Port-Access group (group A) patients had femoro-femoral cannulation with a special arterial cannula to introduce an endoaortic balloon clamp (n = 23). The second group (group B) of patients underwent femoro-femoral CPB as well in combination with a specially designed transthoracic aortic clamp (Chitwood technique, n = 35). Patients were assigned to either technique in a nonrandomized fashion. Demographics were similar in both groups. RESULTS: In group A, 4 valves were replaced, 19 patients had mitral valve repair. In group B, 7 patients had valve replacement and 28 patients underwent mitral repair. Four patients in group A were converted to Chitwood technique due to endoclamp dysfunction. Operating time, CPB time, cross-clamp time, and postoperative blood loss were lower in group B (operating time 295 +/- 83 min vs. 236 +/- 63.9 min; CPB min 167.6 = 64.9 min vs. 137.6 +/- 38.2 min; cross-clamp time 105.9 +/- 51.7 min vs. 78.9 +/- 25.2 min; postoperative blood loss 584 +/- 428 mL vs. 323 +/- 209 mL [p < 0.05]). Clinical outcome regarding postoperative mechanical ventilatilation time, hospital stay and hospital mortality was not different between groups. CONCLUSIONS: Minimally invasive mitral valve procedures via right anterolateral minithoracotomy, including complex valve repair, can be performed successfully using either technique. However, the Chitwood technique provides better intraoperative handling with shorter operation time and less postoperative blood loss. Additionally, costs of a procedure are less using the Chitwood technique compared to the Port-Access technique.

Adolescent↗
PubMed2000

Acute cortisol responses and wound healing in lambs after ring castration plus docking with or without application of a castration clamp to the scrotum.

OBJECTIVE: To test whether the cortisol response to ring castration plus docking is reduced by additional application of a castration clamp across the full width of the scrotum distal to the ring. DESIGN: A physiological study with controls. PROCEDURE: Lambs, 3 to 6 weeks of age, were castrated using a ring or ring plus castration clamp applied for 6 or 10 s and docked using a ring. Blood samples were taken before and regularly for about 4 h after treatment and analysed for plasma cortisol concentrations. The healing of the scrotal wounds was monitored for 6 weeks after castration. RESULTS: The plasma cortisol concentrations were lower only at 60 min after treatment in lambs castrated with a clamp placed on the scrotum for 10 s after ring castration and docking than in lambs castrated and docked by ring alone. Scrotal wounds healed more quickly after ring plus clamp than after ring only castration. CONCLUSIONS: The castration clamp had at most a marginal effect on the cortisol response to ring castration and docking of 3- to 6-week-old lambs, but it did seem to improve the rate of healing.

Animals↗
PubMed1979

Measurement of calcium influx under voltage clamp in molluscan neurones using the metallochromic dye arsenazo III.

1. The metallochromic indicator dye, arsenazo III, was injected into somata of molluscan neurones from Archidoris monteryensis. Membrane current and dye absorbance change were simultaneously monitored under voltage clamp. 2. Absorbance measured at 660 nm increased during positive-going voltage steps large enough to activate membrane conductances. In situ idifference spectra were qualitatively similar to dye, dye-calcium difference spectra recorded in vitro. The absorbance change was abolished by either a thorough removal of external calcium or internal chelation of calcium by EGTA. It was concluded that the absorbance increase primarily reflected changing internal calcium concentration and that the calcium entered from the outside. 3. Dye absorbance increased in a nearly linear fashion during voltage clamp pulses of 100--300 msec duration. This is in qualitative agreement with electrical stuidies which demonstrated only fractional inactivation of calcium conductance during such periods. Plots of absorbance change vs. Vm peaked at +30 to +40 mV and fell off sharply until approximately +70 mV where the slope became less steep. A null or reversal of the absorbance change was generally observed aroung +110 mV. Evidence is presented that calcium influx was in some cases sufficient to cause sizeable changes in its equilibrium potential. 4. During multisecond voltage clamps the slope of the absorbance change showed a large decline. Where barium substituted for calcium as the influx species in identical clamps, the absorbance at 660 nm also increased but in a much more linear fashion. Except for a slight effect on the initial few pulses, the absorbance signal did not recover after a period of barium influx. These results suggest that part of the slope decline might result from processes related to calcium uptake and not to membrane conductance decrease. 5. Dye absorbance changes during normal and TEA action potentials were measured. Comparison of these changes with voltage clamp records indicated that calcium influx during a spike was capable of raising concentration by roughly 2 X 10(-7) M if the cell were considered to be a uniform sphere with no buffering capacity. Calcium influx during action potentials was increased dramatically by TEA, primarily as a result of a prolonged plateau phase. The existence and duration of the plateau was controlled mainly by potassium conductance systems, however. There was no evidence found for facilitation of the calcium conductance. 6. Following a moderate influx of calcium it required 20--60 sec, depending on the neurone, for the dye absorbance to return to base line (at 9 degrees C). The recovery time course showed a marked difference when examined at different wavelengths. For lambda = 660 nm there was an initial period in which the absorbance decreased rapidly, followed by a slower phase which generally carried the absorbance below the initial (prepulse) value...

Action Potentials↗
PubMed1979

A voltage-clamp study of the light response in solitary rods of the tiger salamander.

1. Single, isolated, rod photoreceptors were obtained by enzymatic dissociation of the tiger salamander (Ambystoma tigrinum) retina. These solitary cells retained the morphological features of rods of the intact retina and could be maintained in culture for several days. Solitary cells were penetrated with one or two micropipettes and their electrophysiology was studied by the voltage-clamp technique. 2. Intracellular recording with two micropipettes demonstrated that the inner segment of a solitary rod was effectively isopotential with the outer segment. 3. The time course of the voltage response to a flash resembled that of responses observed in rods in the intact retina. At low light intensities the response reached a peak in approximately 0.7 sec and then slowly declined. At high light intensities the time to peak response decreased and an initial transient arose as the response, after reaching the peak, quickly decreased to a less polarized plateau. 4. The normal voltage response could be compared with the current observed during a voltage clamp. At low light intensities the time course of the current response resembled the time course of the voltage response. When light intensity was increased the time course of the current response differed from the voltage response in that the time to peak amplitude remained relatively constant and an initial transient did not occur. It was possible to predict the current response produced by any intensity of light by using (i) an empirical equation which reproduced the time course of a dim response and (ii) the Michaelis-Menten equation. 5. The time course of the voltage-clamp current produced by a flash was the same at different values of maintained voltage. 6. The maximum amplitude of the voltage-clamp current produced by a flash or step of light was a non-linear function of membrane potential. It was relatively constant within the physiological range, decreased as the membrane potential was moved toward 0 mV, reversed polarity between 0 and 10 mV, and rapidly increased in magnitude as membrane potential was made more positive. Although this current was voltage dependent, no time dependence was evident (recording resolution greater than or equal to 5 msec). 7. Voltage-clamp experiments demonstrated an inward current which slowly developed after a hyperpolarizing voltage step. The effect of this voltage and time dependent current was to reduce, after a delay, the polarization initiated by light.

Ambystoma↗
PubMed1981

Voltage clamp and internal perfusion of single rat heart muscle cells.

1. Single cells from adult rat ventricle were dispersed using an enzymic dissociation technique. Electrical properties were measured with either suction pipettes or conventional glass micropipettes and the results were compared. 2. Suction pipette and micropipette measurements of resting membrane potentials and action potentials were comparable. Values were similar to those reported previously for both dispersed cardiac myocytes and whole tissue preparations from adult rat ventricle. 3. Voltage clamp with a single suction pipette was used in initial experiments, but the results were not sufficiently accurate. Consequently, voltage clamp of single cells was carried out using two suction pipettes (tip diameters 10-15 micrometers), one for passing current and the other for recording membrane potential. Dialysis of cell contents was performed by each suction pipette. A roving micropipette (tip diameter less than 1 micrometer) was used occasionally to measure membrane potential at selected sites. 4. Using the two-suction-pipette method, voltage-clamp steps rose with time constants of less than 10 microsec and the capacitative current transient decayed with a single time constant of less than 100 microsec. These values are more optimal than those observed in other voltage-clamped cardiac muscle preparations. 5. Single cardiac myocytes had membrane input resistances of 44.5 +/- 4.6 M omega, membrane time constants of 16.2 +/- 0.63 msec and membrane capacitances of 399.7 +/- 42.2 pF. (values are means +/- DS, n = 10-). The length constant, lambda, of a single myocte at ists resting potential and perfused with KH2PO4 was about 500 micrometers. For cells perfused with Cs aspartate solution commonly used in voltage-clamp experiments, the input resistance was approximately quadrupled and lambda was 1100 micrometers. The average length of a myocyte partially aspirated by two suction pipettes was 50 micrometers. At maximum -gNa in 50% extracellular Na, lambda was calculated to be about four times the actual cell length between voltage-recording and current-passing pipettes. 6. The half-time for the disappearance or recovery of outward Na currents, following removal or restitution of intracellular Na with two pipettes, was of the order 1 min, indicating that intracellular ionic composition of the cell could be readily controlled and modified.

Action Potentials↗
PubMed1984

Intracellular calcium measured with calcium-sensitive micro-electrodes and Arsenazo III in voltage-clamped Aplysia neurones.

Selected neurones of the abdominal ganglion of Aplysia californica were voltage clamped, injected with the Ca2+-indicator dye Arsenazo III, and impaled with Ca2+-selective micro-electrodes. Measurements of the absorbance signal (Arsenazo III) and Ca2+ micro-electrode potential during and following voltage-dependent Ca2+ influx (induced by voltage-clamp pulses) were simultaneously recorded. In neurones held at -50 mV, the mean intracellular free Ca2+ concentration [( Ca]i) measured by the Ca2+ micro-electrode was 0.18 microM, S.D. = 0.22 microM, n = 13. Bathing the cell in 0 Ca2+ artificial sea water (ASW) or intracellularly injecting EGTA decreased the resting [Ca]i. Voltage-clamp pulses, which maximally activated Ca2+ channels (from -50 to +30 mV), transiently increased both the Arsenazo III absorbance and the Ca2+ micro-electrode signals, indicating a rise in [Ca]i. Given the Ca2+ micro-electrode's limited band width, the peak of the Ca2+ signal during the pulse train could not be resolved; however, there was a net deflexion of this signal following the last pulse which slowly decayed to base line. Bathing the cells in 0 Ca2+ ASW, or reducing the driving force for Ca2+ entry (by stepping the voltage-clamp pulses to much higher membrane potentials) dramatically reduced both the absorbance and the Ca2+ micro-electrode signal increases. On the other hand, bathing the cells in 100 mM-Ca2+ ASW increased both signals. The intracellular Ca2+ gradient within the cytoplasm following voltage-clamp pulses was investigated by moving the Ca2+-selective micro-electrode tip in a step-wise manner relative to the membrane surface. The measured rise in [Ca]i was greatest near the membrane and not measurable within 40-50 microns of the membrane surface. The amplitude of the [Ca]i rise at different distances from the membrane could be fitted by a model based on a simple diffusion of Ca2+ from a plane source.

Action Potentials↗
PubMed2000

Affinity purification of DNA and RNA from environmental samples with peptide nucleic acid clamps.

Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO(4), 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]) a PNA clamp recovered significantly more target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO(4), 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of > or =100 pg were generally >20% but depended upon the specific probe, solution background, and salt condition. The DNA probe had a lower absolute detection limit of 100 fg of target (830 zM [1 zM = 10(-21) M]) in high-salt buffer. In the absence of exogenous DNA (e.g., soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification and detection (830 zM) than the DNA oligomer. In varied environmental samples, the rank order for capture probe performance in high-salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA oligomer generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of nontarget DNA and RNA. Significant interactions between probe type and environmental sample indicate that the most efficacious capture system depends upon the particular sample type (and background nucleic acid concentration), target (DNA or RNA), and detection objective.

Chromatography, Affinity↗
PubMed1992

Carbachol induces K+, Cl-, and nonselective cation conductances in T84 cells: a perforated patch-clamp study.

We used the perforated patch-clamp technique to examine cell membrane ionic conductances in isolated cells of the human colonic secretory cell line, T84, during exposure to the muscarinic agonist carbachol. Carbachol (100 microM) induced both outward and inward currents when the patch pipette contained a normal intracellular-like solution, the bath contained a normal extracellular-like solution, and the cells were intermittently voltage clamped between K+ and Cl- equilibrium potentials. The outward current was identified as a K+ current that averaged 483 +/- 95 pA, while the inward current averaged 152 +/- 29 pA (n = 15). The outward and inward currents oscillated with a synchronous frequency of 0.036 +/- 0.006 Hz; however, the onset of the K+ current occurred an average of 457 +/- 72 ms before the onset of the inward current. When the pipette contained a high-NaCl solution, the bath contained a Na(+)-gluconate solution, and the cells were intermittently voltage clamped between Cl- and Na+ equilibrium potentials, carbachol induced both Cl- and nonselective cation currents. The Cl- current averaged 455 +/- 73 pA, while the nonselective cation current, averaged 336 +/- 54 pA (n = 14). No difference was observed in the onset of these two currents. These results indicate that carbachol induces three separate ionic conductances in T84 cells. We used the whole cell patch-clamp technique in a previous study of these cells [D. C. Devor, S. M. Simasko, and M. E. Duffey. Am. J. Physiol. 258 (Cell Physiol. 27): C318-C326, 1990] and found that carbachol induced only an oscillating membrane K+ conductance. Thus some unidentified component of the carbachol-sensitive signal transduction pathway is diffusible and may be lost during whole cell patch clamping.

Amiloride↗
PubMed1993

Role of angiotensin and adrenoceptors in hemodynamic response to aortic cross-clamping.

An earlier study has shown that angiotensin and catecholamines were responsible for the vasoconstriction observed in the isolated hindlimb preparation during aortic cross-clamping. That study also demonstrated that when vasoconstriction was blocked with an alpha-adrenergic antagonist, phenoxybenzamine, vasodilation was elicited by aortic cross-clamping. The present study tested the hypothesis that this vasodilation was mediated via beta-adrenergic receptors. Eighteen dogs had their hindlimb denervated, vascularly isolated, and pump perfused with blood drained from the inferior vena cava, after passing through a gas-exchanging membrane where oxygen and carbon dioxide tensions were normalized. Left and right thoracotomies were performed, and the aorta and inferior vena cava were cross-clamped. The cross-clamping was associated with 29-37% increase in limb vascular resistance in control dogs (n = 6), in animals pretreated with propranolol (2 mg/kg, n = 6), and in dogs pretreated with a combination of phenoxybenzamine (3 mg/kg) and propranolol (2 mg/kg, n = 6). In animals pretreated with a combination of phenoxybenzamine, propranolol, and enalaprilat (2 mg/kg, n = 6), an angiotensin-converting enzyme inhibitor, limb vascular resistance did not change. This study has confirmed that aortic cross-clamping is associated with vasoconstriction induced by angiotensin and activation of alpha-adrenoceptors and has further demonstrated that vasodilation is attributable to beta-adrenoceptor activation.

Angiotensin II↗