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A new clamp for use in microvascular end-to-side anastomosis is described. The advantages of the clamp are that the end of the donor vessel is placed precisely over the opening of the recipient vessel without tension, good exposure of the posterior wall is provided, and the tension on the stay sutures is easily adjusted.
Albino rats were used to determine the effects of the reapplication of microvascular clamps in veins after anastomosis. The results demonstrate that, if necessary, microvascular clamps can be reapplied either proximal, or both proximal and distal to a venous anastomosis for up to 30 minutes, thus allowing a suture to be redone or the lumen to be explored with no loss of patency.
A key to understanding the mysterious hierarchic organization of annelid multi-subunit extracellular hemoglobin (giant Hb, composed of 144 globin chains and about 36 nonheme chains called linkers) is knowing the role of linkers in holding together the entire two-tiered hexagonal form. Here, the effects of added monosaccharides on the dissociation of giant Hb from the marineworm Perinereis aibuhitensis were monitored using dynamic light scattering (DLS), transmission electron microscopy (TEM), and circular dichroism (CD) measurements. Changes in Stokes radius and more clearly the distribution analysis of the Hb based on the DLS measurements showed that Hb preferentially dissociates into hexagonal units (called submultiples), which was consistent with the results of TEM and CD measurements. The results thus show that linkers specifically "clamp" submultiples together to organize the two-tiered form through carbohydrate gluing. Thus, a submultiple behaves like an ordinary protein, whereas the intact Hb behaves like a miniature supramolecular system. This clamp model is plausible because it inherently involves catastrophe of the molecular stoichiometry at the two-tiered hexagonal formation level because carbohydrates are under posttranslational regulation and therefore contain structural ambiguity.
Apolipoprotein B (apoB) mRNA editing leads to a single base change in its mRNA and the production of apoB-48. Currently, the degree of apoB mRNA editing is analyzed by the RT-PCR primer extension method. While this method is quantitative, it is labor intensive, utilizes radioactivity for labeling and may not be sensitive enough to discriminate between low levels of editing and inherent assay background levels. Peptide nucleic acid (PNA) oligonucletides have been used in single point mutation detection through PCR clamping. In the present work, we developed a PCR based assay which can detect the single base change responsible for the apoB-48 production. We found that as low as 0.5% of the edited form can be clearly detected by PNA mediated PCR clamping. When combined with the primer extension assay, an approximately 180-fold enrichment of the edited percentage is observed, reflecting selected PCR amplification of templates containing the edited base.
Here we report the isolation and characterization of a clamp-loader complex from the thermoacidophilic archaeon Sulfolobus solfataricus (SsoRFC). SsoRFC is a hetero-pentamer composed of polypeptides of 37 kDa (small subunit) and 46 kDa (large subunit), which possess primary structure similarity with human replication factor C p40 and p140 subunits, respectively. The two SsoRFC polypeptides were co-expressed in Escherichia coli and purified as a complex (SsoRFC-complex) that was demonstrated to possess a native M(r) of about 200 kDa and a 4:1 (small to large) subunit stoichiometric ratio. The small subunit was individually expressed in E. coli, purified, and found to form a homo-tetramer (SsoRFC-small; native M(r) 156 kDa), which was also characterized. The SsoRFC-complex, but not SsoRFC-small, highly stimulated the synthetic activity of S. solfataricus B1-type DNA polymerase in reactions containing primed M13mp18 DNA, ATP, and either of the two poliferating cell nuclear antigen-like processivity factors of S. solfataricus (039p and 048p). Both SsoRFC-small and -complex were able to hydrolyze ATP, but only the ATPase activity of the holo-enzymatic assembly was activated by primed DNA templates, such as poly(dA)-oligo(dT). As measured by nitrocellulose filter binding assays, SsoRFC-complex bound poly(dA)-oligo(dT), but not the unprimed homopolymer, whereas SsoRFC-small was devoid of any DNA-binding activity. The peculiar properties of this archaeal clamp-loader complex and their significance for the understanding of the DNA replication process in Archaea are discussed.
The rapid application of caffeine to cardiac myocytes is commonly used to assess changes in the Ca2+ content of the sarcoplasmic reticulum (SR) and to study other parameters of intracellular Ca2+ regulation. Here we examined the effects of rapid caffeine application on membrane potential, intracellular Ca2+, and cell shortening in ventricular myocytes (rat, rabbit, guinea pig, dog) and atrial myocytes (rabbit) that were not voltage clamped. Conditioning pacing was used to achieve a steady-state level of SR Ca2+ loading prior to caffeine (10 mM) application. Caffeine transiently depolarized myocytes as expected from activation of forward Na+-Ca2+ exchange. However, we also found in each species (50% rat, 36% rabbit ventricular, 53% rabbit atrial, 56% guinea pig, 31% dog) that the caffeine-induced depolarization could also trigger an action potential. Caffeine-triggered potentials were completely blocked by thapsigargin (1 microM). The Ca2+ transient and contraction that accompanied caffeine-triggered action potentials had a larger magnitude and slower rate of decline (or relaxation) than occurred during caffeine-induced subthreshold depolarizations. Thus, the use of rapid caffeine application to study SR function and [Ca2+]i regulation in myocytes that are not voltage clamped can yield erroneous results.
The effects of ischemia/reperfusion during 70% partial hepatectomy on hepatic regeneration was evaluated in normal rats and thioacetamide-induced cirrhotic rats. Total hepatic ischemia and reperfusion (15 min each) by portal-triad cross-clamping was repeated two times in normal rats and four times in cirrhotic rats during hepatectomy. The labeling index of hepatocytes on Day 1 after operation (POD) and hepatic regeneration ratio on POD 28 were measured. In normal rats, the repeated ischemia/reperfusion decreased the survival rate from 100 to 77% (P < 0.05), lowered the hepatocyte labeling index from 33.5 +/- 2.5 to 16.7 +/- 6.5%, and diminished the hepatic regeneration ratio from 199 +/- 17 to 137 +/- 12% (P < 0.01). However, portal-systemic shunt improved those levels to 100, 30.6 +/- 13.7, and 169 +/- 19%, respectively (P < 0.01). In cirrhotic rats, no portal congestion was observed and hepatic regeneration was not suppressed by ischemia/reperfusion. Thus, portal pooling and the reperfusion of pooled portal blood may be the cause of inhibition on hepatic regeneration and not ischemia/reperfusion of the liver itself. Therefore, repeated portal-triad cross-clamping for short periods of time during the resection of cirrhotic liver is not harmful for hepatic regeneration.
The effect of piperidine (Pip) on isolated Aplysia neurons was investigated using the voltage clamp and concentration clamp techniques in which neurons were perfused internally and externally with Na+,K+ free solution. Pip induced a Cl-current (ICl) in a dose-dependent manner for doses ranging from 10(-4) M to 10(-2) M. The dose-response curve gave an apparent dissociation constant of 8.4 x 10(-4) M and a Hill coefficient of 1.7. The current-voltage relationship was linear in the voltage range examined (-70 to +30 mV). The equilibrium potential for Pip induced current was close to the calculated equilibrium potential for chloride ions (ECl), (-10.7 mV). The activation phase of the ICl was characterized by a single exponential at all concentrations. The time constant of this phase decreased with increasing concentrations of Pip but did not depend on the membrane potential. The deactivation phase of the ICl proceeded on a single exponential curve at concentrations of Pip less than 5 x 10(-4) M, but on a double exponential at concentrations of 5 x 10(-4) M and higher. The deactivation time constant also decreased with increasing concentrations of Pip, but showed no potential dependence. Pip- and ACh-induced IClS were not blocked by 10(-4) M atropine. However, Pip-induced ICl was abolished with 10(-4) M d-tubocurarine (dTC), and the ACh-induced ICl was depressed by the same dose of dTC. These results suggest that Pip acts on at least two components of the nicotinic receptor-Cl channel complex in Aplysia neurons to elicit the ICl.
Cone photoreceptor inputs to H1 horizontal cells (H1 HCs) in carp retina were studied by measuring light-modulated currents (IL) to monochromatic stimuli (460, 533, 688 nm) under a voltage-clamp condition. By using double-barrelled micro-electrodes H1 HCs were voltage-clamped whilst perfusing with dopamine to uncouple the cells. The IL of the H1 HCs driven by each cone input was segregated by selective chromatic adaptation, and differences in the kinetics of the IL of the H1 HCs were revealed. Thus, all together, three types of IL were observed: (1) a 'fast outward' current to the long-wavelength stimulus; (2) a 'slow outward' current to the middle-wavelength stimulus; and (3) a 'delayed inward' current that followed the peak of 'slow outward' current to the short-wavelength stimulus. The reversal potentials of the three currents were estimated to be at least 20 mV more positive than the dark resting potential by extrapolation of the IL-V curve. These observations are consistent with the idea that the H1 HCs receive sign-inverting, conductance decreasing synaptic input(s) from at least one other cone mechanism, in addition to the main conventional EPSP type synaptic input from red-sensitive cones.
A variant of the whole-cell patch clamp technique is described which allows measurement of whole-cell ionic currents in small cells while minimizing cell dialysis with the pipette solution. The technique involves the application of negative pressure to the inside of small (less than 1 micron) tip diameter pipettes placed on the cell surface to achieve high resistance seals and membrane rupture. The technique has been used successfully in a variety of different types of cells to study membrane currents carried by Ca and K, currents generated by exchange carriers as well as electrical coupling between cells. Overall, the technique seems well suited for the study of ionic currents in small cells, and provides an alternative to conventional patch clamping techniques which necessitate intracellular dialysis.
Two main methods are available for assessing insulin sensitivity with the hyperinsulinaemic euglycaemic clamp technique: one employs a glucose-controlled insulin infusion system (the Biostator) with automatic feedback control; the second depends on frequent glucose measurement and the use of an algorithm and a pocket calculator ('manual') to determine the glucose infusion rate. The amount of glucose infused is a measure of insulin sensitivity. The efficiency of the two methods was compared in nine normal subjects (seven lean, two obese). After an overnight fast subjects were infused with insulin at 50 mU X kg-1 X h-1 for 2 h; this rate was doubled during the first 10 min for the manual technique. Blood glucose averaged 4.7 +/- 0.1 and 4.8 +/- 0.1 mmol/l from 0 to 120 min for Biostator and manual techniques and did not deviate significantly from the desired level. Variability of the clamp was also similar over the same period (coefficient of variation 5.1 +/- 0.6% and 6.4 +/- 0.7%, Biostator and manual). Glucose infused to maintain steady state from 60 to 120 min was higher, however, with the manual than the Biostator method (5.7 +/- 0.6 versus 4.4 +/- 0.6 mg X kg-1 X min-1, p less than 0.01) even when the loading dose was omitted, although the two methods correlated closely (p less than 0.05). Glucose infusion rate varied more from minute to minute with the Biostator (coefficient of variation 28.8 +/- 3% versus 12.2 +/- 2.1%). Steady-state serum insulin levels (30-120 min) were the same during both methods.(ABSTRACT TRUNCATED AT 250 WORDS)
The behavior under voltage clamp conditions of a coupled kinetic scheme for the sodium channel is examined. The scheme is given diagrammatically by: Numerical simulations are presented which show that this model fits the voltage clamp data which are well described by the Hodgkin-Huxley equations, but also gives the sorts of behavior anomalous to the Hodgkin-Huxley model which have been seen experimentally. Further, straightforward changes in parameter values are shown to be capable of mimicking the ways in which some axonal preparations differ from others. Detailed, but admittedly heuristic, arguments are presented for the propositions that: 1) the model is minimal; i.e. no simpler kinetic model will fit the array of data simulated, and: 2) the transient excited state is necessary; i.e. no model of comparable simplicity with pure voltage dependent kinetics will fit the array of data simulated.
The effect of elevated plasma insulin concentration (55 +/- 2 mU/l) on peripheral clearance and production of total ketone bodies was determined using 3-14C-acetoacetate tracer infusions. Nine normal subjects were studied twice, once during insulin infusion (20 mU.m-2.min-1), once during basal plasma insulin concentrations (controls). Blood total ketone body concentrations (sum of acetone, acetoacetate and beta-hydroxybutyrate) were maintained in both studies at 2 mmol/l by feedback-controlled sodium acetoacetate infusions. The coefficient of variation of total ketone body concentrations during the two clamp studies was 10 and 11% respectively. The sodium acetoacetate infusion rate required during the clamp was 55 +/- 4% higher during hyperinsulinaemia than in controls (p less than 0.005). This was due to increased total ketone body clearance (8.4 +/- 0.7 vs 6.7 +/- 0.4 ml.kg-1.min-1, p less than 0.015), and to enhanced suppression of ketone body production (p less than 0.01). Hyperketonaemia alone decreased ketone body production by 42% and diminished ketone body clearance by 46%, the former being enhanced, the latter being in part antagonised by insulin. Since the plasma insulin concentrations were within those observed in patients treated for diabetic ketoacidosis, the data suggest that the antiketotic effect of insulin therapy results in part from an increase in peripheral ketone body disposal.
In an effort to understand the gating properties of ionic channels in biological membranes, an efficient method was developed to estimate the kinetic constants from opening-closing events of the channels. Our method is suitable to single channel patch-clamp recordings that contain several ionic channels functioning simultaneously. It is different from the maximum likelihood method previous developed by Horn and Lange, in that our method is a continuum approach and makes uses of analytic expressions of the probability density functions of the event times. Combinatorial analysis was necessary to correctly include more typical multi-channel recordings. This yields computationally quicker results than the method of Horn and Lange, which uses a discretized time series. Model-dependent portions of the code are minimal and easily modified. To illustrate the goodness of our method, we have generated the open-close processes of the patch-clamp records on a digital computer using the exponential random number generators. For multi-channel patches, we have introduced a few plausible approximations to make our algorithm more efficient. The soundness of the our approximations were tested with such measures as the fraction of the open state at time t, Popen (t), and frequencies of the number of openings per run. A copy of the computer code implementing this algorithm can be obtained from the authors.
Individual ion channels are electrically isolated and studied in living cells with the tight patch voltage clamp method. Channels are identified, categorized, and sometimes named on the basis of the biophysical properties obtained with this method. Although it is usually presumed that these recordings are from native, undisturbed membrane, the physical basis of this technique is not well established. Observations that lipid blebs readily form when suction is applied to patch clamp electrodes suggest that many single channel recordings are from ion channels in these blebs.
Thin slices (100-200 microns) from cardiac ventricles of newborn (1-14 days old) rats were used for patch-clamp recordings. High resistance seals (greater than 10 GOhms) could be obtained on this preparation without enzymatic treatment of tissue. Resting potential for cardiac fibres, measured in whole-cell configuration, ranged between -30 and -65 mV. Averaged sodium currents and inward rectifying potassium single-channel currents, recorded in cell-attached mode, displayed similar features to those previously reported for isolated rat ventricular cells. Application of patch-clamp techniques to cardiac slices allows single-channel recordings without complicated procedures of cell isolation. Moreover, possible alteration of channel properties caused by proteolytic enzymes can be avoided. This technique provides a new tool for studying electrophysiological properties of heart cells in situ.
We have used the patch-clamp technique to characterize three anion channels in the ventricular membrane of the choroid plexus epithelium from Necturus. The most frequently occurring channel had a nonlinear IV-curve. The conductance in excised patches with 112 mM chloride at both sides was 28 pS at 0 mV, increasing towards positive membrane potentials. The selectivity ratios were PNa:PCl less than or equal to 0.1 and PNO3:PCl:PHCO3 = 1.6:1:0.43. SITS and furosemide (1 mM) on the inside reduces chloride flux to 0.15 and 0.37 times the control value. In attached patches, the most commonly observed channel had a conductance of 7.5 pS. The single-channel current for this channel reversed direction at 15 mV hyperpolarization, indicating accumulation of chloride to a factor of 1.8 above equilibrium. External stimulation of the tissue by theophylline, IBMX and dbcAMP, or by hypotonic shock did not increase the activity of this channel. In very few excised patches, we have observed a chloride channel with a conductance of 7 pS with 112 mM chloride at both sides. The 7 pS channel appears to be identical to a 2 pS channel found in attached patches. The 2 pS channel was not normally active in attached patches but was activated in 28% of the patches by external stimulation. Finally, in few excised patches we have found a 375 pS channel which inactivates within seconds when membrane potential is stepped from 0 mV to a value that differs more than 10-20 mV from zero. The channel did not conduct gluconate but PNO3:PCl = 1.08 and PNa:PCl less than or equal to 0.1. Internal SITS and furosemide (1 mM) reduced chloride flux to 0.3 and 0.5 times the control value. The channel was never seen in attached patches. The current carried through these channels can not account for the transepithelial steady state Cl- -flux measured by microelectrodes. KCl exit from the cell is suggested to be carried by KCl-cotransport or by channels that are too small to be seen in patch-clamp experiments.
A macro cell-attached patch-clamp technique using large (16 microns) electrodes was developed to study the properties of the Na current (INa) in the motor endplate region of frog interosseal muscle fibres (resting potential approx.-99 mV). The fibre isolation procedure allows the formation of gigaOhm seals and thus a good voltage-clamp control of the patch. At 22 degrees C, in the presence of 110 mM external [Na] and of K channel blockers, INa activates at -49.6 +/- 1.9 mV, reaches a maximum of 4.33 +/- 0.66 mA/cm2 at -7.5 +/- 2.5 mV and reverses at Vrev equal to +63.2 +/- 1.9 mV. There is no evidence for the presence of a tubular INa. When the [Na] in the recording pipette is changed, Vrev exactly follows the Nernst equation for Na ions. An internal [Na] of 9.69 mM is determined. The Na conductance is maximum (63.56 +/- 7.03 mS/cm2) at +8.9 +/- 3.0 mV and markedly decreases for potentials positive to +25 mV. In contrast, the Na permeability (maximum of 6.13 +/- 0.95 x 10(-4) cm/s at +51.4 +/- 5.6 mV) remains more constant at positive potentials. The Na channels are half-inactivated at -68.9 mV and half-activated at -37.9 mV. At the potential at which INa is maximum, the half-time of activation is 223.9 +/- 10.5 microseconds, the time to peak 365.7 +/- 13.5 microseconds and the time constant of inactivation 260.7 +/- 11.2 microseconds. The time constant of reactivation at -100 mV is 1.44 +/- 0.19 ms. These and other results show that INa can be adequately studied with this technique.