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PubMed2004

Calcium transport by sarcoplasmic reticulum Ca-ATPase can be investigated on a solid-supported membrane.

Sarcoplasmic reticulum (SR) native vesicles incorporating Ca-ATPase are adsorbed on a solid-supported lipid membrane (SSM). Upon adsorption, the ion pumps are chemically activated by concentration jumps of ATP and the capacitive current transients generated by SR Ca-ATPase are measured under potentiostatic conditions. The Michaelis-Menten constant, K(M), for ATP is evaluated by varying the concentration of ATP in the activating solution. This preliminary result shows that ion transport by SR Ca-ATPase can be suitably investigated by a technique based on concentration jumps on an SSM.

Adenosine Triphosphate↗
PubMed2005

ATP and purinergic receptor-dependent membrane traffic in bladder umbrella cells.

The umbrella cells that line the bladder are mechanosensitive, and bladder filling increases the apical surface area of these cells; however, the upstream signals that regulate this process are unknown. Increased pressure stimulated ATP release from the isolated uroepithelium of rabbit bladders, which was blocked by inhibitors of vesicular transport, connexin hemichannels, ABC protein family members, and nucleoside transporters. Pressure-induced increases in membrane capacitance (a measure of apical plasma membrane surface area where 1 microF approximately equals 1 cm2) were inhibited by the serosal, but not mucosal, addition of apyrase or the purinergic receptor antagonist PPADS. Upon addition of purinergic receptor agonists, increased capacitance was observed even in the absence of pressure. Moreover, knockout mice lacking expression of P2X2 and/or P2X3 receptors failed to show increases in apical surface area when exposed to hydrostatic pressure. Treatments that prevented release of Ca2+ from intracellular stores or activation of PKA blocked ATPgammaS-stimulated changes in capacitance. These results indicate that increased hydrostatic pressure stimulates release of ATP from the uroepithelium and that upon binding to P2X and possibly P2Y receptors on the umbrella cell, downstream Ca2+ and PKA second messenger cascades may act to stimulate membrane insertion at the apical pole of these cells.

Adenosine Triphosphate↗
PubMed2004

Disposable noncompetitive immunosensor for free and total prostate-specific antigen based on capacitance measurement.

This work reports on the successful integration of a one-step lateral flow immunoassay format and impedance detection of the specific affinity event using an electrochemical transducer coated with a pH-sensitive polymer layer. This approach was particularly applied to the development of a rapid single-use immunosensor for the sensitive detection of free and total prostate-specific antigen (f-PSA, t-PSA) tumor marker. Strips of nitrocellulose membrane were coated with appropriate antibodies to f-PSA and t-PSA and used as solid supports for the performance of noncompetitive immunoassays where PSA was allowed to react with both immobilized anti-PSA antibody and anti-PSA urease enzyme conjugate for less than 1 min. An additional piece within the device consisting of a storage blister filled with a urea solution allowed the rapid washing of unbound species from the membrane strips and simultaneous urea hydrolysis catalyzed by the bound urease conjugate in an automatic fashion. The hydrolysis of urea increased the pH of the reaction media, which in turn induced a breakdown of the polymer layer on the transducer and a consequent measurable change in capacitance of the system. This was easily recorded at a given frequency over a 30-min period. Overall, we describe a one-step immunosensor prototype that exhibits enough sensitivity to detect both forms of PSA at concentration levels down to 3 ng/mL. With the possibility of being portable and considering its ease of use, robustness, and simplicity, this device has great potential as a tool for the screening and early detection of prostate cancer.

Biosensing Techniques↗
PubMed2005

Vesicle endocytosis requires dynamin-dependent GTP hydrolysis at a fast CNS synapse.

Molecular dependence of vesicular endocytosis was investigated with capacitance measurements at the calyx of Held terminal in brainstem slices. Intraterminal loading of botulinum toxin E revealed that the rapid capacitance transient implicated as "kiss-and-run" was unrelated to transmitter release. The release-related capacitance change decayed with an endocytotic time constant of 10 to 25 seconds, depending on the magnitude of exocytosis. Presynaptic loading of the nonhydrolyzable guanosine 5'-triphosphate (GTP) analog GTPgS or dynamin-1 proline-rich domain peptide abolished endocytosis. These compounds had no immediate effect on exocytosis, but caused a use-dependent rundown of exocytosis. Thus, the guanosine triphosphatase dynamin-1 is indispensable for vesicle endocytosis at this fast central nervous system (CNS) synapse.

Animals↗
PubMedDate not supplied

Hydrating effects of a corticoid oil formulation and its vehicle on human skin.

Factors in the treatment of atopic dermatitis include restoring skin moisture and reducing inflammation. This study evaluated a corticoid oil formulation and its components with respect to their skin hydration potential. Ten healthy Caucasians were enrolled. Five test sites on the left and right forearm of each subject were tested: one site served as a normal skin control (without treatment), whereas four were wetted by spraying distilled water (approximately 0.1 ml) over a 3-cm2 skin surface area, and spraying was repeated every 5 min for a total of three applications. Five minutes after the final application, 0.2 ml of the corticoid oil formulation, moisturizing vehicle, and plain peanut oil were applied to each pre-designated site (3 cm2); one site was kept as a blank control (water saturation only). Thirty minutes later, test sites were gently wiped with paper tissues, and visual scoring, transepidermal water loss (TEWL), and capacitance were recorded and repeated at 2 and 3 h. The corticoid oil formulation, plain peanut oil, and moisturizing vehicle significantly increased skin hydration 30 min after each single application, with no statistically significant difference among the treatments at any point. The corticoid oil formulation and plain peanut oil slightly but not significantly elevated TEWL 30 min after application. The results support intuitive dermatologic judgment of advising patients to apply moisturizing medicaments after bathing.

Adrenal Cortex Hormones↗
PubMed2005

A sensitive immunoassay based on electropolymerized films by capacitance measurements for direct detection of immunospecies.

Fabrication of a capacitive immunosensor based on electropolymerized polytyramine (Pty) film for the direct detection of human serum albumin (HSA) without any labeling is described. The capacitance change of the heterostructures, Pty films/covalently bonded antibodies/buffered medium, is utilized for monitoring the specific antibody-antigen interaction. The Pty films are ultrathin and the HSA assay is nearly specific. Experimental parameters affecting antibody immobilization and the sensing of HSA are investigated in detail and optimized. This capacitive sensor prepared with the present method can provide high sensitivity. Under the optimized experimental conditions, a linear calibration curve in the concentration range 1.84-368.6 ng/ml when plotted vs the logarithm of the antigen concentration is obtained and the detection limit (S/N=3) is 1.60 ng/ml. After an acidic washing the present system can be used again. The applicability and reliability of the sensor are also demonstrated.

Animals↗
PubMed2003

Capacitative calcium entry as a pulmonary specific vasoconstrictor mechanism in small muscular arteries of the rat.

(1) The effect of induction of capacitative Ca2+ entry (CCE) upon tone in small (i.d. 200-500 microm) intrapulmonary (IPA), mesenteric (MA), renal (RA), femoral (FA), and coronary arteries (CA) of the rat was examined. (2) Following incubation of IPA with 100 nm thapsigargin (Thg) in Ca2+-free physiological salt solution (PSS), a sustained contraction was observed upon reintroduction of 1.8 mm Ca2+, which was unaffected by either diltiazem (10 microm) or the reverse mode Na+/Ca2+ antiport inhibitor KB-R7943 (10 microm). An identical protocol failed to elicit contraction in MA, RA, or CA, while a small transient contraction was sometimes observed in FA. (3) The effect of this protocol on the intracellular Ca2+ concentration ([Ca2+]i) was assessed using Fura PE3-loaded IPA, MA, and FA. Reintroduction of Ca2+ into the bath solution following Thg treatment in Ca2+-free PSS caused a large, rapid, and sustained increase in [Ca2+]i in all the three types of artery. (4) 100 nm Thg induced a slowly developing noisy inward current in smooth muscle cells (SMC) isolated from IPA, which was due to an increase in the activity of single channels with a conductance of approximately 30 pS. The current had a reversal potential near 0 mV in normal PSS, and persisted when Ca2+-dependent K+ and Cl- currents were blocked; it was greatly inhibited by 1 microm La3+, 1 microm Gd3+, and the IP3 receptor antagonist 2-APB (75 microm), and by replacement of extracellular cations by NMDG+. (5) In conclusion, depletion of intracellular Ca2+ stores with Thg caused capacitative Ca2+ entry in rat small muscular IPA, MA, and FA. However, a corresponding contraction was observed only in IPA. CCE in IPA was associated with the development of a small La3+- and Gd3+-sensitive current, and an increased Mn2+ quench of Fura PE-3 fluorescence. These results suggest that although CCE occurs in a number of types of small arteries, its coupling to contraction appears to be of particular importance in pulmonary arteries.

Animals↗
PubMed2003

Kinetics of DNA binding with chloroquine phosphate using capacitive sensing method.

The capacitive sensing method has been applied to study the binding of DNA with chloroquine phosphate. DNA was immobilized on a gold electrode surface, self-assembled with thioglycolic acid. The results of a quartz crystal impedance (QCI) study indicate that the reaction of double-strand DNA (dsDNA) with chloroquine includes a fast electrostatic attraction and a slow intercalation of chloroquine into double-strand helix. The real-time experimental data obtained by capacitive sensing also revealed two distinctive kinetics stages during binding of dsDNA with chloroquine, while only one stage exists during reaction of single-strand DNA (ssDNA) with chloroquine. The kinetic parameters were obtained by fitting the real-time experimental data using a two stage reaction model. The rate constants of electrostatic attraction for dsDNA and ssDNA are estimated as 0.014 and 0.018 s(-1), respectively. The rate constant of the second stage of dsDNA is 0.0011 s(-1).

Adsorption↗
PubMed2003

Analysis of hydrostatic pressure-induced changes in umbrella cell surface area.

All cells experience and respond to external mechanical stimuli including shear stress, compression, and hydrostatic pressure. Cellular responses can include changes in exocytic and endocytic traffic. An excellent system to study how extracellular forces govern membrane trafficking events is the bladder umbrella cell, which lines the inner surface of the mammalian urinary bladder. It is hypothesized that umbrella cells modulate their apical plasma membrane surface area in response to hydrostatic pressure. Understanding the mechanics of this process is hampered by the lack of a suitable model system. We describe a pressure chamber that allows one to increase hydrostatic pressure in a physiological manner while using capacitance to monitor real-time changes in the apical surface area of the umbrella cell. It is demonstrated that application of hydrostatic pressure results in an increase in umbrella cell apical surface area and a change in the morphology of umbrella cells from roughly cuboidal to squamous. This process is dependent on increases in cytoplasmic Ca(2+). This system will be useful in further dissecting the mechanotransduction pathways involved in cell shape change and regulation of exocytic and endocytic traffic in umbrella cells.

Animals↗
PubMed2002

Capacitance steps and fusion pores of small and large-dense-core vesicles in nerve terminals.

The vesicles that package neurotransmitters fall into two distinct classes, large dense-core vesicles (LDCVs) and small synaptic vesicles, the coexistence of which is widespread in nerve terminals. High resolution capacitance recording reveals unitary steps proportional to vesicle size. Measurements of capacitance steps during LDCV and secretory granule fusion in endocrine and immune cells have provided important insights into exocytosis; however, extending these measurements to small synaptic vesicles has proven difficult. Here we report single vesicle capacitance steps in posterior pituitary nerve terminals. These nerve terminals contain neuropeptide-laden LDCVs, as well as microvesicles. Microvesicles are similar to synaptic vesicles in size, morphology and molecular composition, but their contents are unknown. Capacitance steps of two characteristic sizes, corresponding with microvesicles and LDCVs, were detected in patches of nerve terminal membrane. Both types of vesicles fuse in response to depolarization-induced Ca(2+) entry. Both undergo a reversible fusion process commonly referred to as 'kiss-and-run', but only rarely. Fusion pores seen during microvesicle kiss-and-run have a conductance of 19 pS, 11 times smaller than LDCV fusion pores. Thus, LDCVs and microvesicles use structurally different intermediates during exocytosis.

Biological Transport↗
PubMedDate not supplied

Characterization and mitigation of stray radiofrequency currents during monopolar resectoscopic electrosurgery.

STUDY OBJECTIVE: To determine patterns and range of stray radiofrequency (RF) currents flowing through the working element of monopolar resectoscopes during routine endometrial rollerball ablation or resection; and to determine whether straightforward modifications of the uterine resectoscope and the application of RF monitoring could provide a safe pathway for such currents. DESIGN: Prospective in vivo measurements (Canadian Task Force classification II-1). SETTING: University-affiliated teaching hospital. PATIENTS: Twelve women undergoing resectoscopic surgery. INTERVENTIONS: During routine resectoscopic surgery using 1.5% glycine irrigant solution, three modified 26F Storz resectoscope working elements (model 27070E) were adapted to be continuously monitored with an Encision AEM device for excessive capacitive coupling and other stray currents from insulation failure. Active electrodes used were 3 mm and 5 mm rollberballs and 8 mm-diameter cutting loops powered by ERBE or Valleylab generators at 120 W. Active and working element currents were monitored by Pearson current transformers followed by root-mean-squared detectors based on the Analog Devices AD-637 integrated circuit. Data were recorded using a Fluke 199C oscilloscope, then serially transferred to a notebook computer and analyzed using Flukeview, Excel, and Minitab software. RESULTS: Typical values of working element currents ranged from 0.10 to 0.20 A. Active electrode currents were typically in the range of 0.50 to 1.10 A. Frequently, the working element current exceeded the typical values and ranged up to 0.60 A. These current surges produced a heat factor (I(2)t) of 0.45 A(2).sec in a 10-second period. CONCLUSIONS: During resectoscopic electrosurgery, baseline, most likely capacitive coupled, currents were always present. In addition, high values of working element currents occurred frequently, and they surged up to 0.60 A for significant periods of time. Without the modification of the resectoscopic device, these currents have the capability of flowing through the patient's genital tract and causing burns. Since monopolar electrosurgery remains an integral part of most hysteroscopic procedures, active electrode monitoring may offer a solution in protecting the patient and the surgeon from stray electrosurgical burns.

Adult↗
PubMed2006

Two modes of fusion pore opening revealed by cell-attached recordings at a synapse.

Fusion of a vesicle with the cell membrane opens a pore that releases transmitter to the extracellular space. The pore can either dilate fully so that the vesicle collapses completely, or close rapidly to generate 'kiss-and-run' fusion. The size of the pore determines the release rate. At synapses, the size of the fusion pore is unclear, 'kiss-and-run' remains controversial, and the ability of 'kiss-and-run' fusion to generate rapid synaptic currents is questionable. Here, by recording fusion pore kinetics during single vesicle fusion, we found both full collapse and 'kiss-and-run' fusion at calyx-type synapses. For full collapse, the initial fusion pore conductance (G(p)) was usually >375 pS and increased rapidly at > or =299 pS ms(-1). 'Kiss-and-run' fusion was seen as a brief capacitance flicker (<2 s) with G(p) >288 pS for most flickers, but within 15-288 pS for the remaining flickers. Large G(p) (>288 pS) might discharge transmitter rapidly and thereby cause rapid synaptic currents, whereas small G(p) might generate slow and small synaptic currents. These results show that 'kiss-and-run' fusion occurs at synapses and that it can generate rapid postsynaptic currents, and suggest that various fusion pore sizes help to control the kinetics and amplitude of synaptic currents.

Animals↗
PubMed2003

Postnatal maturational changes in rat pelvic autonomic ganglion cells: a mixture of steroid-dependent and -independent effects.

Androgens have potent effects on the maturation and maintenance of a number of neural pathways involved in reproductive behaviors in males. Most studies in this area have focused on central pathways, but androgen receptors are expressed by many peripheral neurons innervating reproductive organs, and previous studies have demonstrated structural and chemical changes in these neurons at puberty and after castration. We have performed the first electrophysiological comparison of pelvic autonomic ganglion neurons in male rats before and after puberty and following pre- or postpubertal castration. Studies were performed in vitro on intact ganglia with hypogastric and pelvic nerves attached to allow synaptic activation of sympathetic or parasympathetic neurons, respectively. Pelvic ganglion neurons underwent many changes in their passive and active membrane properties over the pubertal period, and some of these changes were dependent on exposure to circulating androgens. The most pronounced steroid-dependent effects were on membrane capacitance (soma size) in sympathetic neurons and duration of the action potential afterhyperpolarization in tonic neurons. Our study also showed that rat pelvic ganglion cells and their synaptic inputs were more diverse than previously reported. In conclusion, this study demonstrated that rat pelvic ganglion neurons undergo considerable postnatal changes in their electrophysiological properties. The steroid dependence of some of these changes indicates that circulating androgens may influence reproductive behaviors at many locations within the nervous system not just in the brain and spinal cord.

Age Factors↗
PubMed2003

Ultrathin alumina sol-gel-derived films: allowing direct detection of the liver fibrosis markers by capacitance measurement.

A capacitive immunoassay based on antibody-embedded ultrathin gamma-alumina sol-gel films (approximately 20 to 40 nm) was successfully prepared in this work. The nanofilms greatly increased the capacitance change initiated by the recognition between the immobilized antibody and the target antigen, which allowed capacitive measurements capable of directly determining the antigen more sensitive than that of thick films. Meanwhile, the inorganic films with high permittivity significantly increased the time constant (i.e., RC value) of the films, which rendered the potentiostatic step method with acceptable S/N ratio. These two advantages enabled the immunosensor to be readily employed in a multichannel capacitance analysis system. An eight-channel hIgG capacitive sol-gel-derived immunoassay based on this system was constructed to illustrate the application. Compared with the detection limits of SiO2 sol-gel-derived hIgG capacitive immunosensors or the conventional ELISA immunoassay, the immunoassay based on thin alumina gel film showed a lower detection limit of 1 ng mL(-1). The novel immunoassay was employed to co-determine two liver fibrosis markers (hyaluronan and laminin) in mixed samples from approximately 0.5 to 50 ng mL(-1). The little derivation caused by the interfered antigen indicated that the sensitive, specific, low-cost sol-gel-derived multichannel immunosensors might be a promising approach in the application of screening disease markers.

Aluminum Oxide↗
PubMed2003

Endothelin-1 stimulated capacitative Ca2+ entry through ET(A) receptors of a rat brain-derived type-1 astrocyte cell line, IA-1g1.

The present study demonstrated that endotheline-1 (ET-1) stimulated a biphasic (transient and sustained) increase in [Ca(2+)](i) and signaling was blocked by BQ123 and inhibited by BQ788. RT-PCR analysis revealed that ET(A) was expressed more than ET(B) mRNA-suggesting that ET(A) is the major receptor. Simply reintroducing Ca(2+) in the buffer stimulated a sustained increase in [Ca(2+)](i) and the effect was inhibited by U73122, thapsigargin (TG), miconazole and SKF96365. When measured in Ca(2+)-free buffer, the ET-1-stimulated Ca(2+) transient decreased by 73% and the reintroduction of Ca(2+) induced a large sustained increase in [Ca(2+)](i). These effects were not affected by nifedipine, but were inhibited by miconazole and SKF96365-indicating that the sustained increase in [Ca(2+)](i) mediated by ET-1 was mostly due to capacitative Ca(2+) entry (CCE). The ET-1-induced CCE was inhibited by phorbol ester (PMA) but was enhanced by GF109203X; it was also enhanced by 8-bromo-cyclic AMP (8-Br-cAMP) but was inhibited by H89. Thus, protein kinase C (PKC) negatively regulated and cAMP-dependent protein kinase (PKA) positively regulated the ET-1-mediated CCE in these cells.

Amino Acids, Cyclic↗
PubMed2006

A micro-scale multi-frequency reactance measurement technique to detect bacterial growth at low bio-particle concentrations.

The technique described enables the user to detect the presence and proliferation of bacteria through an increase in the bulk capacitance (C) of the suspension, which is proportional to the bacteria count, at practical frequencies less than 1 MHz. The geometry of the micro-capillary design employed increases the bulk resistance (R) of the medium, thus increasing its RC time. This makes the measured reactance sensitive to changes in the bulk capacitance, which is usually masked by the much larger surface capacitance. The sensitivity is further enhanced by the existence of a minimum in the value of the reactance at a frequency proportional to the inverse medium RC time. The value of this reactance minimum and the frequency at which the minimum is recorded are dependent on the bacteria count and permit the detection of an initial concentration of approximately 100 CFU ml(-1) of E. coli within 3 hours of incubation, in comparison with the previous reported values of about 8 hours, with an initial load of 1000 CFU ml(-1).

Bacteriological Techniques↗
PubMed2006

NMDA receptors trigger neurosecretion of 5-HT within dorsal raphe nucleus of the rat in the absence of action potential firing.

Activity and calcium-dependent release of neurotransmitters from the somatodendritic compartment is an important signalling mechanism between neurones throughout the brain. NMDA receptors and vesicles filled with neurotransmitters occur in close proximity in many brain areas. It is unknown whether calcium influx through these receptors can trigger the release of somatodendritic vesicles directly, or whether postsynaptic action potential firing is necessary for release of these vesicles. Here we addressed this question by studying local release of serotonin (5-HT) from dorsal raphé nucleus (DRN) neurones. We performed capacitance measurements to monitor the secretion of vesicles in giant soma patches, in response to short depolarizations and action potential waveforms. Amperometric measurements confirmed that secreted vesicles contained 5-HT. Surprisingly, two-photon imaging of DRN neurones in slices revealed that dendritic calcium concentration changes in response to somatic firing were restricted to proximal dendritic areas. This implied that alternative calcium entry pathways may dominate the induction of vesicle secretion from distal dendrites. In line with this, transient NMDA receptor activation, in the absence of action potential firing, was sufficient to induce capacitance changes. By monitoring GABAergic transmission onto DRN 5-HT neurones in slices, we show that endogenous NMDA receptor activation, in the absence of postsynaptic firing, induced release of 5-HT, which in turn increased the frequency of GABAergic inputs through activation of 5-HT(2) receptors. We propose here that calcium influx through NMDA receptors can directly induce postsynaptic 5-HT release from DRN neurones, which in turn may facilitate GABAergic input onto these cells.

Action Potentials↗
PubMed2006

Instrumental assessment of atopic eczema: validation of transepidermal water loss, stratum corneum hydration, erythema, scaling, and edema.

BACKGROUND: Quantification of skin diseases can be carried out in many ways. Clinical scores are widely used in atopic eczema (AE), and noninvasive instruments are a relevant supplement. OBJECTIVE: Our purpose was to validate 5 noninvasive instruments in quantification of AE severity. METHODS: In all, 101 patients with AE and 30 control subjects were assessed twice in a clinical cross-sectional examination. Assessment of transepidermal water loss, stratum corneum hydration, erythema, scaling, and subepidermal edema was assessed on 3 predetermined skin sites. RESULTS: The methods discriminated among various severity degrees and correlated significantly with objective assessment of disease severity. High correlations were found among instruments assessing acute symptoms of AE. Threshold values for transepidermal water loss and capacitance were found. LIMITATIONS: No gold standard exists for severity assessment of atopic eczema. Therefore, the methods used cannot be validated in relation to such a standard. Furthermore, atopic eczema is a generalized disease and the methods used assess target lesions. By assessing target lesions, information about the disease is reduced. CONCLUSION: Noninvasive instruments are valuable in quantification of disease severity in a mixed group of patients with active AE. Assessment with ultrasound has contributed new information about the pathophysiology in AE.

Adolescent↗