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At least 1,765 records · Page 98Linked to original sources
PubMed2004

Exendin-4 pharmacodynamics: insights from the hyperglycemic clamp technique.

The purpose of this study is to ascertain the pharmacodynamic properties of exendin-4, a glucose-dependent insulinotropic agent, from plasma glucose and insulin concentration-time profiles following a 60-min intravenous infusion in healthy and type 2 diabetic subjects. Plasma glucose and insulin concentrations were obtained from a previous clinical study, whereby a hyperglycemic clamp was established and maintained in healthy (n = 7) and type 2 diabetic (n = 7) volunteers (plasma glucose raised 5.4 mM above fasting level). Exendin-4 was infused (0.15 pmol/kg/min) during the 2nd hour of a 5-h clamp. A physiological pharmacodynamic model was developed and fitted to individual glucose and insulin responses simultaneously. Because drug concentrations were unavailable, hypothetical pharmacokinetic driving functions were approximated during the modeling process and used to enhance a proportionality constant relating elevated glucose and the rate of second-phase insulin release. Exendin-4 infusions produced substantial insulin release in both subject populations that required higher glucose infusion rates to maintain stable hyperglycemia. Observed plasma glucose-insulin profiles were well characterized by the final pharmacodynamic model. Apparent exendin-4 elimination rate constants for healthy and diabetic subjects were similar (0.0386 +/- 0.0192 and 0.0460 +/- 0.0145 min(-1)). Capacity and sensitivity parameters of drug effect were 2-fold lower in diabetic subjects, but mean differences were not statistically significant. Simulations confirm that diabetic subjects exhibit a reduced capacity to enhance second-phase insulin release in response to exendin-4 compared with healthy subjects. Type 2 diabetic subjects demonstrate a significant response to exendin-4, but to a lesser extent than nondiabetic subjects, despite comparable measures of apparent drug exposure and efficacy.

Diabetes Mellitus, Type 2↗
PubMed2005

A phenylalanine clamp catalyzes protein translocation through the anthrax toxin pore.

The protective antigen component of anthrax toxin forms a homoheptameric pore in the endosomal membrane, creating a narrow passageway for the enzymatic components of the toxin to enter the cytosol. We found that, during conversion of the heptameric precursor to the pore, the seven phenylalanine-427 residues converged within the lumen, generating a radially symmetric heptad of solvent-exposed aromatic rings. This "phi-clamp" structure was required for protein translocation and comprised the major conductance-blocking site for hydrophobic drugs and model cations. We conclude that the phi clamp serves a chaperone-like function, interacting with hydrophobic sequences presented by the protein substrate as it unfolds during translocation.

Amino Acid Sequence↗
PubMed2006

A clamping mechanism involved in SNARE-dependent exocytosis.

During neurotransmitter release at the synapse, influx of calcium ions stimulates the release of neurotransmitter. However, the mechanism by which synaptic vesicle fusion is coupled to calcium has been unclear, despite the identification of both the core fusion machinery [soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)] and the principal calcium sensor (synaptotagmin). Here, we describe what may represent a basic principle of the coupling mechanism: a reversible clamping protein (complexin) that can freeze the SNAREpin, an assembled fusion-competent intermediate en route to fusion. When calcium binds to the calcium sensor synaptotagmin, the clamp would then be released. SNARE proteins, and key regulators like synaptotagmin and complexin, can be ectopically expressed on the cell surface. Cells expressing such "flipped" synaptic SNAREs fuse constitutively, but when we coexpressed complexin, fusion was blocked. Adding back calcium triggered fusion from this intermediate in the presence of synaptotagmin.

Adaptor Proteins, Vesicular Transport↗
PubMed1976

Extracellular potassium accumulation and inward-going potassium rectification in voltage clamped ventricular muscle.

Measurements of afterpotential, action potential duration, and output of a potassium-sensitive microelectrode indicate that the application of long clamp pulses (1 to 8 seconds) to frog ventricular muscle is accompanied by a change in the extracellular potassium concentration. The plot of the magnitude of the potassium accumulation against the clamped membrane potential yields an N-shaped relation similar to the "steady state" current-voltage relation. The accumulation studies confirm a strong inward-going (anomalous) potassium rectification.

Action Potentials↗
PubMed1967

Giant axon of Myxicola: some membrane properties as observed under voltage clamp.

A new space-voltage clamped giant (500 to 900 microns) axon preparation is described (Myxicola infundibulum). Normal value for resting and action potentials are 71 and 89 millivolts, respectively. This preparation under voltage clamp exhibits relations between current and voltage like those described for the squid axon. The early inward current component is reduced in a solution with low sodium concentration. This preparation, then, acts in all its essential features like the squid giant axon. Myxicola, however, can be made available the year around and should prove to be an extremely useful preparation for the study of excitable membranes.

Animals↗
PubMed1970

Changes in extrinsic fluorescence in squid axons during voltage-clamp.

Fluorescence changes in squid axons were examined after staining with rhodamine B, pyronin B, or 8-anilinonaphthalene-1-sulfonate by intracellular application. Gradual changes in fluorescence were detected during both hyperpolarizing and depolarizing voltage-clamp pulses. Abrupt changes were often observed at the onset and at the end of voltage-clamp. Possible sources of artifact in optical measurements of this type and some implications of the findings are discussed.

Animals↗
PubMed1989

Regulation of calcium concentration in voltage-clamped smooth muscle cells.

The regulation of intracellular calcium concentration in single smooth muscle cells was investigated by simultaneously monitoring electrical events at the surface membrane and calcium concentration in the cytosol. Cytosolic calcium concentration rose rapidly during an action potential or during a voltage-clamp pulse that elicited calcium current; a train of voltage-clamp pulses caused further increases in the calcium concentration up to a limit of approximately 1 microM. The decline of the calcium concentration back to resting levels occurred at rates that varied with the calcium concentration in an apparently saturable manner. Moreover, the rate of decline at any given calcium concentration was enhanced after a higher, more prolonged increase of calcium. The process responsible for this enhancement persisted for many seconds after the calcium concentration returned to resting levels. Thus, the magnitude and duration of a calcium transient appear to regulate the subsequent calcium removal.

Action Potentials↗
PubMed1981

Voltage clamp studies in macrophages from mouse spleen cultures.

Voltage clamp studies of macrophages from cultures of mouse spleen macrophages produced N-shaped steady-state current-voltage curves containing a region of negative slope resistance. Some macrophages exhibit two stable states of membrane potential, having current-voltage relationships that cross the voltage axis at three points. Outward currents that turn on at voltages of +15 millivolts or greater were noted in several cells. The addition of barium chloride to the bathing medium abolished the negative slope resistance and reduced the inward currents in response to hyperpolarizing voltage steps. These data provide direct evidence that macrophages exhibit at least tow different voltage-dependent conductances and demonstrate that voltage clamp techniques can be useful in studying the membrane properties of leukocytes.

Animals↗
PubMed2006

The plasmid RK2 replication initiator protein (TrfA) binds to the sliding clamp beta subunit of DNA polymerase III: implication for the toxicity of a peptide derived from the amino-terminal portion of 33-kilodalton TrfA.

The broad-host-range plasmid RK2 is capable of replication and stable maintenance within a wide range of gram-negative bacterial hosts. It encodes the essential replication initiation protein TrfA, which binds to the host initiation protein, DnaA, at the plasmid origin of replication (oriV). There are two versions of the TrfA protein, 44 and 33 kDa, resulting from alternate in-frame translational starts. We have shown that the smaller protein, TrfA-33, and its 64-residue amino-terminal peptide (designated T1) physically interact with the Escherichia coli beta sliding clamp (beta(2)). This interaction appears to be mediated through a QLSLF peptide motif located near the amino-terminal end of TrfA-33 and T1, which is identical to the previously described eubacterial clamp-binding consensus motif. T1 forms a stable complex with beta(2) and was found to inhibit plasmid RK2 replication in vitro. This specific interaction between T1 and beta(2) and the ability of T1 to block DNA replication have implications for the previously reported cell lethality caused by overproduction of T1. The toxicity of T1 was suppressed when wild-type T1 was replaced with mutant T1, carrying an LF deletion in the beta-binding motif. Previously, T1 toxicity has been shown to be suppressed by Hda, an intermediate regulatory protein which helps prevent over-initiation in E. coli through its interaction with the initiator protein, DnaA, and beta(2). Our results support a model in which T1 toxicity is caused by T1 binding to beta(2), especially when T1 is overexpressed, preventing beta(2) from interacting with host replication proteins such as Hda during the early events of chromosome replication.

Amino Acid Sequence↗
PubMed1983

A voltage clamp inhibits chemotaxis of Spirochaeta aurantia.

Anaerobic conditions were employed to study the relationship between membrane potential and chemotaxis in Spirochaeta aurantia. When cells were grown anaerobically and suspended in anaerobic potassium phosphate buffer (pH 5.5), membranes did not appear to be polarized. Nevertheless, motility was supported by a transmembrane pH gradient, and the anaerobic cells exhibited D-xylose taxis. Introduction of trace amounts of air into anaerobic cell suspensions resulted in a transient membrane polarization. The addition of valinomycin to cells suspended under anaerobic conditions did not alter the steady-state value of membrane potential appreciably but served to clamp membrane potential at the preset level. Although there was no detectable effect of valinomycin on the motility of anaerobic cells in potassium phosphate buffer, D-xylose taxis was completely inhibited by this treatment. These data indicate the the action of valinomycin as a voltage clamp serves to inhibit the chemotaxis of S. aurantia and provide evidence to support the suggestion that the mechanism of chemotaxis in this organism involves the transduction of sensory signals in the form of membrane potential fluctuations.

Anaerobiosis↗
PubMed1995

DNA hybridization and contour-clamped homogeneous electric field electrophoresis for identification of enterococci to the species level.

In this study, 113 Enterococcus faecium, 37 Enterococcus faecalis, 24 Enterococcus gallinarum, 15 Enterococcus raffinosus, and 13 Enterococcus casseliflavus clinical isolates and American Type Culture Collection (ATCC) strains were evaluated by contour-clamped homogeneous electric field electrophoresis. Thirty-one of the E. faecium, 22 of the E. faecalis, 24 of the E. gallinarum, 15 of the E. raffinosus, and 13 of the E. casseliflavus isolates were also evaluated by DNA-DNA hybridization. Genomic DNAs from type strains E. faecalis ATCC 19433, E. faecium ATCC 19434, E. gallinarum ATCC 49573, E. raffinosus ATCC 49427, and E. casseliflavus ATCC 25788 were labeled with biotin for use as probes. E. faecalis differed from all other species in always having a largest fragment of > 400 kb. E. gallinarum was different from all other species in having all SmaI fragments of < 200 kb. Biotin-labeled probes showed a high degree of hybridization with genomic DNA from the same species and a low degree of hybridization when hybridized to genomic DNA from different species for all isolates tested except for four isolates identified as E. faecium by conventional biochemical methods. The DNA from these four isolates hybridized strongly to DNA from E. gallinarum ATCC 49573 and weakly to E. faecium ATCC 19434 DNA and had all SmaI fragments of < 200 kb in size. These data suggest that these isolates are nonmotile E. gallinarum. DNA from each ATCC type strain hybridized strongly with itself and had only a low degree of hybridization with DNA from other ATCC type strains tested. These results suggest that contour-clamped homogeneous electric field electrophoresis patterns and DNA-DNA hybridization with biotin-labeled probes may be of use for species differentiation of some enterococci.

Blotting, Southern↗
PubMed1997

The effect of Tn916 insertions on contour-clamped homogeneous electrophoresis patterns of Enterococcus faecalis.

Contour-clamped homogeneous gel electrophoresis has increasingly been used and has generally been considered the method of choice for the delineation of enterococcial strains. It has been suggested that the contour-clamped homogeneous electric field (CHEF) electrophoresis digestion patterns of genomic DNA indicate the relatedness of strains when SmaI patterns differ by six or fewer bands. To evaluate the potential reasons for the diversity of bands among clonally related isolates, we studied plasmid-free Enterococcus faecalis FA2-2 and derivatives with transposon Tn916 (encoding for tetracycline resistance) insertions on the chromosome. We obtained derivatives with seven different Tn916 insertion sites and up to two copies of Tn916 on the chromosome, resulting in differences of one to seven bands by CHEF electrophoresis; eight different patterns were observed. With Tn916 insertions, there was either (i) loss of a band(s) with the generation of a new band(s), (ii) the generation of a new band(s) only, or (iii) the loss of a band(s) with no new visible band(s). These results indicate that strains can have up to seven different SmaI bands by CHEF electrophoresis and still be closely related.

Bacterial Typing Techniques↗
PubMed1993

Umbilical cord clamping and preterm infants: a randomised trial.

OBJECTIVE: To investigate the clinical effects of regulating umbilical cord clamping in preterm infants. DESIGN: A prospective randomised study. SETTING: The Queen Mother's Hospital, Glasgow. SUBJECTS: 36 vaginally delivered infants over 27 and under 33 weeks' gestation. INTERVENTION: Holding the infant 20 cm below the introitus for 30 seconds before clamping the umbilical cord ("regulated" group, 17 patients), or conventional management ("random" group, 19 patients). MAIN OUTCOME MEASURES: Initial packed cell volume, peak serum bilirubin concentrations, red cell transfusion requirements, and respiratory impairment (assessed by ventilatory requirements, arterial-alveolar oxygen tension ratio over the first day in ventilated infants, and duration of dependence on supplemental oxygen). RESULTS: There were statistically significant differences between the two groups in mean initial packed cell volume (regulated group 0.564, random group 0.509) and median red cell transfusion requirements (regulated group zero, random group 23 ml/kg). 13 infants from each group underwent mechanical ventilation and showed significant differences in mean minimum arterial-alveolar oxygen tension ratio on the first day (regulated group 0.42, random group 0.22) and in median duration of dependence on supplemental oxygen (regulated group three days, random group 10 days). Differences in final outcome measures such as duration of supplemental oxygen dependence and red cell transfusion requirements were mediated primarily through arterial-alveolar oxygen tension ratio and also packed cell volume. CONCLUSIONS: This intervention at preterm deliveries produces clinical and economic benefits.

Bilirubin↗
PubMed1982

The toxic effects of ouabain: a voltage-clamp study.

The electrophysiologic effects of a toxic concentration of ouabain (10(-5) M) were studied in frog atrial trabeculae. The toxic concentration was determined by the appearance of a negative inotropic effect and an increase in basal tension. Current- and voltage-clamp measurements were performed. Ouabain did not alter the passive electrical properties of the preparation. Under current-clamp conditions the membrane depolarized and the action potential amplitude as well as its maximum rate of rise decreased. The current--voltage curve for the fast inward current was shifted toward more positive potentials and the maximum sodium current decreased. The maximum sodium conductance was also reduced. The process of reactivation of the fast inward current was accelerated. The slow inward current and the maximum slow conductance also decreased under ouabain. These effects could explain the negative inotropic action of high concentrations of glycosides, as well as the action potential changes observed by several investigators. They also help to understand the arrhythmogenic effects of high concentrations of digitalis.

Animals↗
PubMed1990

Patch clamp studies of lymphocyte activation.

In both T- and B-lymphocyte activation, antigen receptor or mitogen stimulation results in phosphoinositide turnover, generation of InsP3 and diacylglycerol, and a sustained rise in intracellular Ca2+, from both intracellular Ca2+ stores release and enhanced transmembrane Ca2+ influx. There is also an associated K+ efflux and membrane hyperpolarization. Patch clamp studies in T and B cells have revealed the presence of several types of ion channels that apparently contribute to the ion fluxes and to the membrane potential changes associated with lymphocyte activation. Three types of T-cell channels are described in this review. First, patch clamp studies have revealed the presence of a nonvoltage-gated, Ca2+ permeable channel, the probability of whose opening increases upon exposure of the T cell to activating ligands. Enhanced opening probability appears to be mediated by the second messenger InsP3, implying that InsP3 is responsible for both intracellular Ca2+ stores release and enhanced transmembrane Ca2+ influx. Thus, the control of [Ca2+]i remains coupled to TCR/CD3 function. The Ca2+ permeable channel also undergoes a Ca2(+)-dependent inactivation process in an autoregulatory fashion. In addition, voltage-gated K+ channels, which closely resemble the delayed rectifier K+ channel of nerve and muscle, can be classified into three subtypes, according to their voltage dependence of activation, inactivation kinetics, and pharmacological sensitivity. The expression of the three K+ channel subtypes varies with the cell's developmental state and functional class. The voltage-activated K+ channel is postulated to have a role in mitogenesis, based on studies that demonstrate an increase in K+ channel amplitude in the 24-48 hr following mitogen stimulation, and on studies that demonstrate that K+ channel blockers inhibit mitogenesis in a dose-dependent manner with the same potency sequence for ion channel block. The precise functional role of the voltage-activated K+ channel remains to be determined. Finally, a Ca2(+)-activated K+ channel in T cells has recently been described. This channel presumably underlies the K+ efflux and membrane hyperpolarization that accompany the mitogen-induced increase in [Ca2+]i. Three channel types that may contribute to activation have also been described in B lymphocytes. In murine myeloma and hybridoma cells, a voltage-gated Ca2+ channel similar to Ca2+ channels in nerve, heart, and muscle is present. It is unclear whether or not this type of Ca2+ channel is present in straight B-cell lines.(ABSTRACT TRUNCATED AT 400 WORDS)

B-Lymphocytes↗
PubMed1998

Cranial bone flap fixation clamps: compatibility at MR imaging.

Metallic cranial bone flap fixation clamps were tested for compatibility at 1.5-T magnetic resonance (MR) imaging with standardized techniques. There was no magnetic field attraction, heating was minor, and artifacts involved a relatively small signal void. These data indicate that bone flap fixation clamps will not present risks to patients undergoing MR imaging procedures with MR systems of 1.5 T or less.

Artifacts↗
PubMed1985

Delay of desensitization onset by potassium ion in voltage-clamped frog muscle fibers.

Increase in extracellular K+ concentration causes delay in desensitization onset during prolonged application of carbamylcholine to the postjunctional membrane in muscle. This could be due to a direct action of K+ on acetylcholine receptors or to some change in the receptors related to K+-induced effects on transmembrane potential. The question of direct vs. voltage-dependent action of K+ was investigated in frog muscle (Rana pipiens) using a point-source voltage clamp. In conductance measurements first without voltage control, desensitization rate in bath media containing 33 mM K+ was -0.198 s-1 among fibers showing an average potential of -30 mV and -0.104 s-1 in 165 mM K+ where the average potential was -2 mV, a decrease of 47%. By comparison, in voltage-clamp tests at a nominal holding potential of +20 mV, increasing extracellular K+ from 33 to 165 mM caused a decrease of 61% in desensitization rate from -0.151 to -0.059 s-1. Another series in 165 mM K+ at a holding level of +10 mV showed a decrease of 67% to a rate of 0.047 s-1. It is concluded that increases in extracellular K+ can delay desensitization onset independently of effects on transmembrane potential. It is suggested that this could result from a direct interaction of K+ with sites on the outer receptor moiety or within channels, but probably not at the inner membrane face, if the latter are considered in equilibrium with bulk intracellular K+.

Animals↗
PubMed1984

Pregnancy-induced insulin resistance in the rat: assessment by glucose clamp technique.

To quantify and characterize the insulin resistance during pregnancy in the rat, a euglycemic hyperinsulinemic clamp was set up. Dose-response curves for the effects of five concentrations of insulin on glucose production, glucose utilization, and glucose clearance were performed in age-matched virgin and 19-day-pregnant rats. Glucose production and glucose utilization were measured by using [3-3H]-glucose. Glucose production was totally suppressed at plasma insulin concentrations higher than 1,000 microU/ml in the two groups. Insulin concentration causing half-maximal suppression of glucose production was about 70 microU/ml in virgin rats and 250 microU/ml in pregnant rats. Maximal glucose utilization was obtained at plasma insulin concentrations of 2,000 microU/ml. In pregnant rats maximal increment in glucose utilization was significantly lower (P less than 0.01) than in virgin rats. Insulin concentrations causing half-maximal stimulation of glucose utilization were 200 microU/ml in virgin rats and 500 in pregnant rats. As blood glucose concentration in virgin and pregnant rats was clamped at, respectively, 0.97 +/- 0.03 and 0.73 +/- 0.03 mg/ml, glucose clearance rates were calculated because this parameter is minimally affected by the changes in blood glucose concentrations. A normal maximal increment in glucose clearance in response to insulin was restored in pregnant rats but the rightward shift of the dose-response curve was maintained. Plasma insulin concentrations necessary for half-maximal increment of glucose clearance in the two groups were similar to that observed when the results were expressed as glucose utilization. Thus, insulin resistance during late pregnancy in the rat is characterized by a decreased sensitivity of liver and peripheral tissues to insulin.

Animals↗