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Poly(binaphthyl-20-crown-6) as receptor based molecular selective potentiometric electrodes for catecholamines and other 1,2-dihydroxybenzene derivatives.

A novel type of poly(crown ether) electrode that is capable of selectively determining some 1,2-dihydroxybenzenes has been developed. A lipophilic macrocyclic crown ether, binaphthyl-20-crown-6, is electrochemically deposited on a platinum disc electrode. The film obtained is used as a sensory element for a potentiometric electrode for the determination of some neurotransmitters, namely, catecholamines. The new electrode is also capable of discriminating the steric shapes of 1,2-dihydroxybenzene moieties. The response of the new electrode is based on the principle of 'host-guest' chemistry. The potentiometric response is dependent on the pH of the solution and the nature of the buffer medium. The new sensor electrode has a useful analytical range of 1.5 x 10(-8) M-2 x 10(-5) M with a linear dynamic range between about 1 x 10(-7) M-5 x 10(-4) M with a 'super-Nernstian' slope of 110-130 mV/decade. The detection limit in phosphate buffer (0.1 M, pH 9.4) is ca. 3 x 10(-8) M for catecholamine. The sensor electrode is virtually insensitive towards interference from most inorganic ions and circumvents the interference from ascorbic acid, which is often found using amperometric methods in biological samples. A partial response mechanism of the present electrode is discussed, supported by results of electron dispersive analysis by x-rays (EDAX).

Animals↗

Ultrasonic masker clarifies ultrasonic perception in man.

Bone conduction enables ultrasound to be heard. Although several hypotheses about ultrasonic perception have been presented, the perception mechanism of bone-conducted ultrasound has not yet been established. In this study, to investigate ultrasonic perception, the amount of masking produced by 27-, 30- and 33-kHz bone-conducted ultrasonic maskers for air-conducted high-frequency sounds was measured in the frequency range of 8-18 kHz at 1-kHz intervals. The results showed that the air-conducted signals in the frequency range of 10-14 kHz were strongly masked by the ultrasonic maskers. When the masker intensity increased from 5 to 10 dB SL, the growth of masking was more than 10 dB in the frequency range of 9-15 kHz, and the masking spread strongly to lower frequencies. Furthermore, the dynamic range for bone-conducted ultrasound was clearly narrower than that for air-conducted high-frequency sounds. These results suggest that perception of bone-conducted ultrasound depends on inner hair cell activity induced by ultrasound, even without modulation being present, and does not depend on enhancement by the outer hair cells in the basal turn of the cochlea.

Adult↗

Cross-correlation of optical microcavity biosensor response with immobilized enzyme activity. Insights into biosensor sensitivity.

Porous silicon multilayer structures have remarkable optical and morphological properties that can be exploited for biosensing. In particular, a high internal surface area (>100 m(2)/cm(3)) and a linear response profile to changes in the dielectric environment enable fabrication of sensitive devices and a straightforward quantitation of the optical response. These essential operating characteristics are illustrated for p+ mesoporous silicon (pore diameter 15-20 nm) optical microcavities. A series of devices were prepared to permit the immobilization of glutathione-S-transferase ( approximately 50 kDa) within the porous matrix. Enzyme activity was exploited as an indirect means to quantitate the amount of protein immobilized. Activity was positively correlated with the optical sensor response. However, at high enzyme load the activity becomes nonlinear while the microcavity response remains linear. These data were used to determine the transduction limit (minimum amount of protein required to transduce an optical response), which is reported as areal mass sensitivity ranging between 50 and 250 pg/mm(2). This value is considered in context with the dynamic range of the bulk sensitivity, defined as the magnitude of the wavelength shift per refractive index unit, which was measured as a function of microcavity design parameters. This work has uncovered key parameters that can be tuned to improve the detection limit of this sensor modality. Because of the ever increasing number of emerging new biosensor technologies, defining sensor detection limits has become an ambiguous topic and a need exists to standardize measurements and sensitivity units. For chip-based devices, it seems appropriate to report sensitivity in terms of the minimum number of grams of bound target per surface area.

Biosensing Techniques↗

[Visualization and quantification of large bowel motility with functional cine-MRI].

PURPOSE: To develop and evaluate a method to visualize and quantify large bowel motility using functional cine MRI. METHODS: Fifteen healthy individuals (8 males, 7 females, 20 to 45 years old) with no history or present symptoms of bowel disorders were enrolled in a functional cine MRI examination at 6 a. m. after a starving phase for at least eight hours before and after oral administration of Senna tea (mild stimulating purgative). Two consecutive sets of repeated measurements of the entire abdomen were performed using a 1.5 T MRI system with coronal T2-weighted HASTE sequences anatomically adjusted to the course of the large bowel. A navigator technique was used for respiratory gating at the level of the right dorsal diaphragm. The changes in diameter (given in cm) were measured at 5 different locations of the ascending (AC), transverse (TC) and descending colon (DC), and assessed as parameters for the bowel motility. RESULTS: The mean values as a statistical measure for large bowel relaxation were determined. Before ingestion of Senna tea, the mean diameter measured 3.41 cm (ascending colon), 3 cm (transverse colon) and 2.67 cm (descending colon). After the ingestion of Senna tea, the mean diameter increased to 3.69 cm (ascending colon) to 3.4 cm (transverse colon) and to 2.9 cm (descending colon). A statistically significant difference was demonstrated with the Wilcoxon test (level of confidence 0.05). For the determination of dynamic increase, the changes of the statistical scatter amplitude to the mean value were expressed as percentage before and after the ingestion of Senna tea. Thereby, an increase in variation and dynamic range was detected for the AC (112.9 %) and DC (100 %), but a decrease in the dynamics for the TC (69 %). CONCLUSION: A non-invasive method for the assessment of bowel motility was developed for the first time. The use of functional cine MRI utilizing a prokinetic stimulus allowed visualisation and quantification of large bowel motility. Further studies have to determine whether this technique is clinically relevant.

Adult↗

Time-resolved fluoroimmunoassay of human choriogonadotropin.

We describe time-resolved fluoroimmunoassay for human choriogonadotropin involving monoclonal antibodies directed against the beta- and alpha-subunits. The latter antibody was labeled with europium, which was measured by counting for 1 s after the immunoreaction was completed. In the solid-phase sandwich assay, both a one-step and two-step procedure were used; the respective measuring ranges were 0.7-135 and 0.7-350 int. units/L, the latter covering a 500-fold dynamic range. The CV within the assay range was between 4 and 8%, depending on the dose. Cross reactivity with lutropin in the one- and two-step procedures was 1.6% and 1.0%, respectively.

Antibodies, Monoclonal↗

Application of a commercially-manufactured Doppler-shift laser velocimeter to the measurement of basilar-membrane vibration.

A commercially-available laser Doppler-shift velocimeter has been coupled to a compound microscope equipped with ultra-long-working-distance objectives for the purpose of measuring basilar membrane vibrations in the chinchilla. The animal preparation is nearly identical to that used in our laboratory for similar measurements using the Mössbauer technique. The vibrometer head is mounted on the third tube of the microscope's trinocular head and its laser beam is focused on high-refractive-index glass microbeads (10-30 microns) previously dropped, through the perilymph of scala tympani, on the basilar membrane. For equal sampling times, overall sensitivity of the laser velocimetry system is at least one order of magnitude greater than usually attained using the Mössbauer technique. However, the most important advantage of laser-velocimetry vis-à-vis the Mössbauer technique is its linearity, which permits undistorted recording of signals over a wide velocity range. Thus, for example, we have measured basilar-membrane responses to clicks whose waveforms have dynamic ranges exceeding 60 dB.

Acoustic Stimulation↗

The primary visual pathway in humans is regulated according to long-term light exposure through the action of a nonclassical photopigment.

BACKGROUND: The mammalian eye shows marked adaptations to time of day. Some of these modifications are not acute responses to short-term light exposure but rely upon assessments of the photic environment made over several hours. In the past, all attempts at a mechanistic understanding have assumed that these adaptations originate with light detection by one or other of the classical photoreceptor cells (rods or cones). However, previous work has demonstrated that the mammalian eye contains non-rod, non-cone photoreceptors. This study aimed to determine whether such photoreceptors contribute to retinal adaptation. RESULTS: In the human retina, second-order processing of signals originating in cones takes significantly longer at night than during the day. Long-term light exposure at night is capable of reversing this effect. Here, we employed the cone ERG as a tool to examine the properties of the irradiance measurement pathway driving this reversal. Our findings indicate that this pathway (1) integrates irradiance measures over time periods ranging from at least 15 to 120 min; (2) responds to relatively bright light, having a dynamic range almost entirely outside the sensitivity of rods; (3) acts on the cone pathway primarily through a local retinal mechanism; and (4) detects light via an opsin:vitamin A photopigment (lambda(max) approximately 483 nm). CONCLUSIONS: A photopigment with a spectral sensitivity profile quite different from those of the classical rod and cone opsins but matching the standard profile of an opsin:vitamin A-based pigment drives adaptations of the human primary cone visual pathway according to time of day.

Adult↗

Highly cooperative control of endocytosis by clathrin.

Clathrin assembles into a dynamic two-dimensional lattice on the plasma membrane where it plays a critical role in endocytosis. To probe the regulation of this process, we used siRNA against clathrin, in combination with single cell assays for transferrin uptake as well as total internal reflection microscopy, to examine how endocytic rates and membrane dynamics depend upon cellular clathrin concentration ([Clathrin]). We find that endocytosis is tightly controlled by [Clathrin] over a very narrow dynamic range such that small changes in [Clathrin] can lead to large changes in endocytic rates, indicative of a highly cooperative process (apparent Hill coefficient, n > 6). The number of clathrin assemblies at the cell surface was invariant over a wide range of [Clathrin]; however, both the amount of clathrin in each assembly and the subsequent membrane dynamics were steeply dependent on [Clathrin]. Thus clathrin controls the structural dynamics of membrane internalization via a strongly cooperative process. We used this analysis to show that one important regulator of endocytosis, the actin cytoskeleton, acts noncompetitively as a modulator of clathrin function.

Actins↗

Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs.

There has been a surge of interest in the biology of microRNAs and the technology of RNA interference. We describe a simple, robust, inexpensive assay for quantitative analysis of microRNAs and short-interfering RNAs. The method relies on primer extension conversion of RNA to cDNA by reverse transcription followed by quantitative, real-time PCR. Technical parameters critical to the success of the assay are presented. Measurements of microRNA levels are sensitive, with most assays allowing measurements in the femtomolar range, which corresponds to tens of copies per cell or less. The assay has a high dynamic range and provides linear readout over differences in microRNA concentrations that span 6-7 orders of magnitude. The assay is capable of discriminating between related microRNA family members that differ by subtle sequence differences. We used the method for quantitative analysis of six microRNAs across 12 tissue samples. The data confirm striking variation in the patterns of expression of these noncoding regulatory RNAs.

DNA Primers↗

[Advanced Dynamic Flow]

There are problems for contrast imaging using LevovistTM contrast agent. In case of Doppler mode, poor resolution and large blooming area are the problems. In case of 2nd harmonic imaging and pulse inversion imaging, tissue harmonic image(THI) becomes the problem because Levovist needs high mechanical index(MI). THI increases under the high MI condition and THI interferes with the contrast image of Levovist. And also bubbles of Levovist are easily collapsed under the high MI condition, so it is hard to visualize the bubbles successively. Dynamic Flow has solved the above problems. Dynamic Flow uses Doppler technique, but Dynamic Flow image has higher resolution and less blooming area than the conventional Doppler image. Dynamic Flow doesn't display THI, so the detectability of bubbles is superior to the other mode as far as Levovist. Dynamic Flow is so sensitive that it can show the bubbles in real time. Advanced Dynamic Flow(ADF) has greatly improved in sensitivity and presentation ability from conventional Dynamic Flow. In the contrast application, ADF image looks very similar to B-mode image thanks to the high resolution and wide dynamic range, in spite of using Doppler technique. Detectability of the bubbles is much better than conventional Dynamic Flow. ADF can detect bubbles in much smaller vessels and has much more penetration, with uniform enhancement from shallow to deep regions. ADF is very suitable for Levovist. ADF can also be used without contrast agents. ADF can show small tiny vessels with high resolution and high frame rate. Directional information can be added by different colors. The sensitivity is close to conventional Color/Power Doppler images.

Journal Article↗

Pulse code modulation telemetry in ski injury research. I. Instrumentation.

Measurement problems can be classified into instrumentation, data transmission and recording, and analysis. This paper focuses on the transmission of multichannel, high-volume, high-frequency, high-accuracy data. Boot-ski dynamometer and skier velocity anemometer data provide 13 channels of max. 8-mV signals requiring 8-microvolt resolution or 4.45-Newton dynamometer resolution. The data transmission system features durability, power consumption approx. 10 Watts, weight 4.54 kp, range greater than 3,500 m, frequency response 250 Hz, accuracy 1 per cent, temperature stability, dynamic range plus or minus 2 inches. The transducer signals are ampflified to plus or minus 10 V for the 100-kbps PCM system. Special AC amplifiers, driven by an amplitude-stabilized power oscillator, were designed for elimination of radio frequency interference (RFI), improved stability and high signal/noise. Sixteen words are sequentially sampled at 521/sec-13 data, 2 frame counters, and 1 sync. The ground station consists of the PCM decoder with real-time capability and an analog tape recorder. Data is subsequently buffered and formatted onto digital tape by mini-computer.

Athletic Injuries↗

Analysis of dicamba degradation by Pseudomonas maltophilia using high-performance capillary electrophoresis.

A method based on high-performance capillary electrophoresis (HPCE) was developed for the simultaneous analysis of dicamba and its metabolites in media containing the bacterium Pseudomonas maltophilia. Dicamba, 3,6-dichlorosalicylic acid (DCSA), and related products were extracted from media samples using ether under acidic conditions and injected onto an HPCE system containing a pH 10.0 running buffer. Baseline resolution between these compounds was obtained at 30 kV with a total run time of 6 min on a 50-microns i.d. x 50-cm capillary. The linear range for dicamba and DCSA detected at 274 nm extended from 0 to 100 mg/liter and the dynamic range for both compounds extended to 2000 mg/liter. The within-run precision was +/- 5% or less throughout the entire concentration range studied. The limits of detection for dicamba and DCSA in the media samples were 6 and 2 mg/liter. This corresponded to detection limits of 0.3 and 0.1 ng, respectively, in the injected extracts. With this method it was possible to conduct preliminary studies examining the kinetics of dicamba metabolism in P. maltophilia.

Capillary Action↗

Peripheral and spinal components of the sensitization of spinal neurons during an acute experimental arthritis.

In cats the injections of kaolin and carrageenan into the knee joint lead to an acute arthritis which develops within 1-3 hours. In parallel articular afferents (low, high threshold and unresponsive ones) are becoming (more) sensitive to movements in the working range of the joint and many show (enhanced) ongoing discharges. Consequently spinal nociceptive-specific and wide dynamic range neurons with afferent input from the inflamed knee develop (increased) responsiveness to gentle stimulation of the joint. But in addition most of these neurons display enhanced reactions to non-inflamed parts of their receptive fields, too, and some neurons show enlargement of their total receptive fields. These latter findings indicate that the sensitization of spinal neurons is not simply reflecting the increased afferent input from the inflamed knee but that intrinsic spinal mechanisms may participate in the sensitization process.

Animals↗

A low-voltage wide-input CMOS comparator for sensor application using back-gate technique.

In this paper, two new analog CMOS comparators (Type-I and Type-II) with low-voltage and wide-input capabilities are proposed. The comparator receives two analog inputs and puts out one digital state to identify the larger (or the smaller) of the input variables, which represents an useful operation in data conversion and sensory signal processing. Without using special fabrication technologies, the supply voltage of the circuit is reduced to 1 V. Due to the utilization of CMOS back-gate technique, the input range of the comparators is greatly improved. The comparators are composed of bulk-driven stage and dynamic latch. By using a CMOS n-well technology, the results of HSPICE simulations indicate that the response time of Type-I circuit is 1 micros under 10 mV identified resolution. Type-II comparator achieves 5 mV identified resolution. The input dynamic ranges of the comparators are approximately rail-to-rail.

Analog-Digital Conversion↗

The adsorption mechanism of nortryptiline on C18-bonded Discovery.

The adsorption isotherms of an ionizable compound, nortriptyline, were accurately measured by frontal analysis (FA) on a C(18)-Discovery column, first without buffer (in an aqueous solution of acetonitrile at 15%, v/v of ACN), then with a buffer (in 28%, v/v ACN solution). The buffers were aqueous solutions containing 20 mM of formic acid or a phosphate buffer at pH 2.70. The linear range of the isotherm could not be reached with the non-buffered mobile phase using a dynamic range larger than 40,000 (from 1.2 x 10(-3) g/L to 50 g/L). With a 20 mM buffer in the liquid phase, the isotherm is linear for concentrations of nortriptyline inferior to 10(-3) g/L (or 3 micromol/L). The adsorption energy distribution (AED) was calculated to determine the heterogeneity of the adsorption process. AED and FA were consistent and lead to a trimodal distribution. A tri-Moreau and a tri-Langmuir isotherm models accounted the best for the adsorption of nortriptyline without and with buffer, respectively. The nature of the buffer affects significantly the middle-energy sites while the properties of the lowest and highest of the three types of energy sites are almost unchanged. The desorption profiles of nortriptyline show some anomalies in relation with the formation of a complex multilayer adsorbed phase of acetonitrile whose excess isotherm was measured by the minor disturbance method. The C(18)-Discovery column has about the same total saturation capacity, around 200 g of nortriptyline per liter of adsorbent (or 116 mg/g), with or without buffer. About 98-99% of the available surface consists in low energy sites. The coexistence of these different types of sites on the surface solves the McCalley's enigma, that the column efficiency begins to drop rapidly when the analyte concentration reaches values that are almost one hundred times lower than those that could be predicted from the isotherm data acquired under the same experimental conditions. Due to the presence of some relatively rare high energy sites, the largest part of the saturation capacity is not practically useful.

Acetonitriles↗

On-line pervaporation-capillary electrophoresis for the determination of volatile analytes in food slurries.

Pervaporation has been coupled on-line to capillary electrophoresis (CE) by a flow injection manifold and the replenishment system of the CE instrument. The approach allows volatile analytes to be removed, derivatisated and injected into the capillary meanwhile the sample matrix remains in the pervaporator. Acetone and four aldehydes (namely: formaldehyde, acetaldehyde, hexenal, 2-trans-hexenal) have been simultaneously determined in slurries samples by this approach. The detection limits (LOD) ranged between 0.1 and 0.6 microg/ml, the quantification limits between 0.5 and 2.0 microg/ml and the linear dynamic ranges between the limit of quantitation and 150 microg/ml. The precision, expressed as relative standard deviation (RSD), ranged between 0.76 and 4.21% for repeatability and between 1.12 and 4.78% for within laboratory intermediary precision. The errors involved in the analysis of the target analytes--expressed as RSD for all compounds--ranged between 0.13 and 4.87%. The optimal pervaporation time and that necessary for the individual separation/detection of the target analytes are 15 and 10 min, respectively. The analysis frequency is 4 h(-1). The accuracy of the method and potential matrix effects were established by analysing spiked samples. Recoveries between 96.12 and 105.67% were obtained. The proposed method was applied to 10 samples with different solid contents (namely, such yoghurt, juice and yoghurt-juice mixtures).

Acetone↗

A combined yeast/bacteria two-hybrid system: development and evaluation.

Two-hybrid screening is a standard method used to identify and characterize protein-protein interactions and has become an integral component of many proteomic investigations. The two-hybrid system was initially developed using yeast as a host organism. However, bacterial two-hybrid systems have also become common laboratory tools and are preferred in some circumstances, although yeast and bacterial two-hybrid systems have never been directly compared. We describe here the development of a unified yeast and bacterial two-hybrid system in which a single bait expression plasmid is used in both organismal milieus. We use a series of leucine zipper fusion proteins of known affinities to compare interaction detection using both systems. Although both two-hybrid systems detected interactions within a comparable range of interaction affinities, each demonstrated unique advantages. The yeast system produced quantitative readout over a greater dynamic range than that observed with bacteria. However, the phenomenon of "autoactivation" by baits was less of a problem in the bacterial system than in the yeast. Both systems identified physiological interactors for a library screen with a cI-Ras test bait; however, non-identical interactors were obtained in yeast and bacterial screens. The ability to rapidly shift between yeast and bacterial systems provided by these new reagents should provide a marked advantage for two-hybrid investigations. In addition, the modified expression vectors we describe in this report should be useful for any application requiring facile expression of a protein of interest in both yeast and bacteria.

Amino Acid Sequence↗

Microarray as a model for quantitative visualization chemistry.

For visualization of proteins or nucleic acids, direct and indirect in situ fluorescence and absorption methods (immunohistochemistry and cytochemistry) have existed for many years. The authors describe a new experimental approach using microarray as a model to quantitatively compare both visualization methods. The spots obtained with the microarray robot had a progressive twofold decrease in concentrations and are used as objects with known amounts of DNA. Subsequent hybridization resulted in a direct fluorescence (DF) label or in hapten for indirect fluorescence (IF) and absorption. The results show that the image of the object in the IF method is larger than that in the DF method because of an edge effect, with stronger staining at the circumference. This leads to a higher plateau level and an 8- to 10-fold reduction in the detection threshold for IF compared with DF. These features are especially useful for one-color DNA-related microarray analysis, such as single nucleotide polymorphism, loss of heterozygosity, and mutation analysis, provided that the spots are not designed directly adjacent to each other, so that the edge effect is taken into account. The slope of the linear range for the IF method is much steeper than for the DF method, pointing to a narrow dynamic range in immunohistochemistry. It is noteworthy that the detection limit for absorption images after indirect immunoenzyme visualization is lower than for the DF images. The indirect immunohistochemistry semiquantitative absorption signal was at least similar compared with the DF fluorescence. In conclusion, an explanation for the difficulties experienced in quantitative immunohistochemistry is provided, and the data emphasize that in general, for daily pathology, semiquantitative patterns should suffice. Indirect labeling of DNA has useful characteristics for application in microarray analyses because of the large signal enhancement.

Carbocyanines↗