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PubMed1995

Storage-phosphor autoradiography: a rapid and highly sensitive method for spatial imaging and quantitation of radioisotopes.

This study evaluates a storage-phosphor imaging system for rapid autoradiography and quantitation of beta- and gamma-ray emitters in mice. Known quantities of authentic 3H, 14C, and 195mPt (0.06 to 31,714 DPM/mm2) were exposed to imaging plates of the storage-phosphor system for 0.5 min to 15 hr at room temperature. Immediately after exposure, the imaging plates were scanned and the autoradiograms were quantified. Radioactivity as low as 1.8 DPM/mm2 (3H), 0.06 DPM/mm2 (14C) and 3.1 DPM/mm2 (195mPt) was visualized and quantified in 8 hr (3H and 14C) and 6 min (195mPt), respectively. The system displayed a linear range of four to five orders of magnitude (14C: 0.5 to 6,914 DPM/mm2 for 2-hr exposure; 3H: 1.8 to 15,372 DPM/mm2 for 15-hr exposure; and 195mPt: 66 to 198,000 DPM 6 min-exposure). Variation from scan-to-scan was small (< 1% to 5%) and was more pronounced at the lower detection limits and exposure periods. Similar sensitivity, linearity, and variability were obtained in cryosections of murine tumors treated with 14C-fluorouracil or 3H-epinephrine and whole-body autoradiograms of mice treated with 195mPt-cisplatin via liver. The high sensitivity allowed noninvasive imaging of live unanesthetized mice treated with 195mPt with a short exposure time (105 sec). These results validate, for the first time, the high sensitivity, linearity, and wide dynamic range of storage-phosphor technology for quantitative autoradiography of 14C, 3H, and 195mPt isotopes. Storage-phosphor imaging will be useful in rapid quantitative autoradiographic disposition studies of radiolabeled drugs and excipients in vitro and in vivo.

Animals↗
PubMed1975

A proposed blind routine quality control system for multichannel analyzers.

A blind quality control system using a special type of commercial reference serum is described. As packaged, the product contains six pools of analyzed reference sera with vials identified by coded numbers. The test constituents in the pools cover the range of multichannel analyzers in a random manner, i.e., each pool contains constituents in the low, normal and elevated ranges. As specific pools are not identified, and the analyst is unaware of the expected values for each constituent, results collected for quality control purposes are unbiased, i.e., free of conscious or unconscious editing. Data analysis in the proposed system is by least-squares technics. During an eight-week period, performance was studied using the standard error of the estimate to evaluate precision and the slope of the regression line to evaluate accuracy on the SMA and Mark X instruments. As an empirical comparison, the data are expressed as relative errors and precision and accuracy of analyzer performance evaluated as mean and standard deviation. The proposed regression analysis-based technic has the advantage of simultaneously evaluating linearity, as well as checking accuracy and precision over the total dynamic range of the instrument by use of a series of related serum pools.

Alkaline Phosphatase↗
PubMed2005

Efficacy of a cochlear implant simultaneous analog stimulation strategy coupled with a monopolar electrode configuration.

OBJECTIVES: The present study was performed to evaluate the efficacy and clinical feasibility of using monopolar stimulation with the Clarion Simultaneous Analog Stimulation (SAS) strategy in patients with cochlear implants. METHODS: Speech recognition by 10 Clarion cochlear implant users was evaluated by means of 4 different speech processing strategy/electrode configuration combinations; ie, SAS and Continuous Interleaved Sampling (CIS) strategies were each used with monopolar (MP) and bipolar (BP) electrode configurations. The test measures included consonants, vowels, consonant-nucleus-consonant words, and Hearing in Noise Test sentences with a +10 dB signal-to-noise ratio. Additionally, subjective judgments of sound quality were obtained for each strategy/configuration combination. RESULTS: All subjects but 1 demonstrated open-set speech recognition with the SAS/MP combination. The group mean Hearing in Noise Test sentence score for the SAS/MP combination was 31.6% (range, 0% to 92%) correct, as compared to 25.0%, 46.7%, and 37.8% correct for the CIS/BP, CIS/MP, and SAS/BP combinations, respectively. Intersubject variability was high, and there were no significant differences in mean speech recognition scores or mean preference ratings among the 4 strategy/configuration combinations tested. Individually, the best speech recognition performance was with the subject's everyday strategy/configuration combination in 72% of the applicable cases. If the everyday strategy was excluded from the analysis, the subjects performed best with the SAS/MP combination in 37.5% of the remaining cases. CONCLUSIONS: The SAS processing strategy with an MP electrode configuration gave reasonable speech recognition in most subjects, even though subjects had minimal previous experience with this strategy/configuration combination. The SAS/MP combination might be particularly appropriate for patients for whom a full dynamic range of electrical hearing could not be achieved with a BP configuration.

Adult↗
PubMed1998

Ion/ion reactions for oligopeptide mixture analysis: application to mixtures comprised of 0.5-100 kDa components.

Oligopeptide mixtures have been subjected to electrospray ionization, accumulated within a quadrupole ion trap, and subjected to ion/ion proton transfer reactions with anions derived from perfluoro-1,3-dimethylcyclohexane. Various mixtures were studied with approximate molecular weight ranges of 0.5-8.5, 12-30, 45-100, and 0.5-100 kDa. Mixtures of known composition were studied to evaluate the mixture complexity amenable to electrospray combined with ion/ion reactions to reduce spectral complexity associated with multiple charging. Mixture analysis with at least 40 components of low and medium molecular weight and roughly comparable solution concentrations appears to be straightforward. No matrix effects upon ionization were implicated in the data for the low and medium molecular weight mixtures but bovine albumin appeared to inhibit signals from bovine transferrin and chicken conalbumin in the high molecular weight mix. Furthermore, the presence of abundant low mass-to-charge ions appeared to inhibit signals from high molecular weight proteins (> 40 kDa) in the 0.5-100 kDa mix. Such an observation is consistent with dynamic range limitations that can arise from discrimination based on ion space charge effects, although an ionization matrix effect could not be precluded from the data reported here. The results reported here indicate that the limitation to mixture complexity amenable to electrospray mass spectrometry imposed by spectral congestion associated with multiple charging can be significantly reduced via ion/ion reactions. The use of ion/ion reactions can therefore facilitate the study of other factors that can impose limitations to mixture analysis, such as matrix effects upon ionization and differences in ion transmission, accumulation, storage, and detection efficiencies.

Algorithms↗
PubMed2002

Fluorescence data analysis on gel-based biochips.

A series of biochip readers developed for gel-based biochips includes three imaging models and a novel nonimaging biochip scanner. The imaging readers, ranging from a research-grade versatile reader to a simple portable one, use wide-field objectives and 12-bit digital large-coupled device cameras for parallel addressing of multiple array elements. This feature is valuable for monitoring the kinetics of sample interaction with immobilized probes. Depending on the model and the label used, the sensitivity of these readers approaches 0.3 amol of a labeled sample per gel element. In the selective scanner, both the spot size of the excitation laser beam and the detector field of view match the size of the biochip array elements so that the whole row of the array can be read in a single scan. The portable version reads 50-mm long, 150-element, one-dimensional arrays in 5 s. With a dynamic range of 4000:1, a sensitivity of 1-5 amol of a labeled sample per gel element, and a data format facilitating online processing, the scanner is an attractive, inexpensive solution for biomedical diagnostics. Fluorophores for sample labeling were compared experimentally in terms of detection sensitivity, influence on duplex stability, and suitability for multilabel analysis and thermodynamic studies. Texas Red and tetracarboxyphenylporphyn proved to be the best choice for two-wavelength analysis using the imaging readers.

Fluorescent Dyes↗
PubMed2006

T-cell antigen-receptor stoichiometry: pre-clustering for sensitivity.

The T-cell antigen receptor (TCR x CD3) is a multi-subunit complex that is responsible for triggering an adaptive immune response. It shows high specificity and sensitivity, while having a low affinity for the ligand. Furthermore, T cells respond to antigen over a wide concentration range. The stoichiometry and architecture of TCR x CD3 in the membrane have been under intense scrutiny because they might be the key to explaining its paradoxical properties. This review highlights new evidence that TCR x CD3 is found on intact unstimulated T cells in a monovalent form (one ligand-binding site per receptor) as well as in several distinct multivalent forms. This is in contrast to the TCR x CD3 stoichiometries determined by several biochemical means; however, these data can be explained by the effects of different detergents on the integrity of the receptor. Here, we discuss a model in which the multivalent receptors are important for the detection of low concentrations of ligand and therefore confer sensitivity, whereas the co-expressed monovalent TCR x CD3s allow a wide dynamic range.

Animals↗
PubMed1988

Eighth nerve fiber firing features in normal-hearing rabbits.

Neural activity of single eighth nerve fibers was recorded with glass microelectrodes in anesthetized normal-hearing rabbits. The units had a spontaneous rate ranging from 0 to approximately 120 spikes/s. In a large number of fibers this rate was below 2 spikes/s. The frequency tuning curves (FTCs) had a tip and a tail region for fibers with a high or medium characteristic frequency (CF). For low-frequency units the FTC was more symmetrically U-shaped. The tip threshold reached the behavioral threshold and units with thresholds of up to more than 60 dB above the mean behavioral threshold were found. There was a weak negative correlation between spontaneous rate and tip threshold. Frequency selectivity, Q10, was about 2 for units with CF below 2 kHz and about 5 for those with CF above 4 kHz. The peristimulus time (PST) histogram showed an initial peak, a plateau, and poststimulus inhibition. For the majority of fibers the dynamic range was 20-30 dB. Some fibers did not reach saturation within the stimulus intensity available. The tip-to-tail distance was 50 dB for high-frequency units at one octave below CF, a matter of potential interest for further studies of animals with inner ear lesions.

Animals↗
PubMed2005

Toxin screening in phytoplankton: detection and quantitation using MALDI triple quadrupole mass spectrometry.

The investigation of a MALDI triple quadrupole instrument for the analysis of spirolide toxins in phytoplankton samples is described in this study. A high-frequency (kHz) laser was employed for MALDI, generating a semicontinuous ion beam, thus taking advantage of the high duty cycle obtained in sensitive triple quadrupole MRM experiments. Initially, several experimental parameters such as type of organic matrix and concentration, solvent composition, and matrix-to-analyte ratio were optimized, and their impact on sensitivity and precision of the obtained ion currents for a reference spirolide, 13-desmethyl-C, was studied. In all quantitative experiments, excellent linearities in the concentration range between 0.01 and 1.75 microg/mL were obtained, with R2 values of 0.99 or higher. The average precision of the quantitative MALDI measurements was 7.4+/-2.4% RSD. No systematic errors were apparent with this method as shown by a direct comparison to an electrospray LC/MS/MS method. Most importantly, the MALDI technique was very fast; each sample spot was analyzed in less than 5 s as compared to several minutes with the electrospray assay. To demonstrate the potential of the MALDI triple quadrupole method, its application to quantitative analysis in several different phytoplankton samples was investigated, including crude extracts and samples from mass-triggered fractionation experiments. 13-Desmethyl spirolide C was successfully quantified in these complex samples at concentration levels from 0.05 to 90.4 microg/mL (prior to dilution to have samples fall within the dynamic range of the method) without extensive sample preparation steps. The versatility of the MALDI triple quadrupole method was also exhibited for the identification of unknown spirolide analogues. Through the use of dedicated linked scan functions such as precursor ion and neutral loss scans, several spirolide compounds were tentatively identified directly from the crude extract, without the usual time-consuming chromatographic preseparation steps. Moreover, high-quality CID spectra were obtained for low-abundant spirolides present in the phytoplankton samples.

Animals↗
PubMed1998

Detection of and discrimination between total and free human interleukin-4 and free soluble interleukin-4 receptor by ELISA.

Interleukin-4 (IL-4) signaling is initiated by binding of IL-4 to the high-affinity IL-4 receptor alpha-chain and subsequent interaction with the common gamma-chain. Soluble forms of the extracellular domain of the alpha-chain (sIL-4R) were shown to be present in biological fluids and, dependent on the concentration, enhance or inhibit IL-4 activity by forming IL-4/sIL-4R complexes. To discriminate between free and potentially active IL-4 from the inactive and complexed form, we have established a set of new ELISA systems for the measurement of human IL-4 in its distinct forms. To select suitable pairs of anti-IL-4 antibodies, a chequerboard interference analysis with six highly-selective human IL-4 specific monoclonal antibodies was performed. For the determination of total IL-4, a monoclonal capture antibody was used that binds IL-4 outside the binding site of the IL-4R alpha-chain. Another antibody recognizing an epitope of the alpha-chain binding site was chosen for the detection of free IL-4. The binding of this antibody was inhibited in a dose-dependent fashion by recombinant sIL-4R. Assays for both total and free IL-4 exhibited a sensitivity of 8 pg/ml and a dynamic range up to 1000 pg/ml. Human sIL-4R was detected by two monoclonal antibodies directed against different epitopes. This ELISA was inhibited by recombinant IL-4 suggesting the measurement of predominantly free sIL-4R. Complexes between soluble IL-4R and IL-4 were detected by a monoclonal anti-sIL-4R antibody in combination with an anti-IL-4 antibody. When supernatants of activated T cells were analyzed, the majority of the IL-4 was in free form. The amount of complexed IL-4 was low as indicated by the fact that most of total IL-4 could be detected as free IL-4. Although values obtained for complexed IL-4 correlated with the difference between total and free IL-4, precise values could not be determined, presumably due to the dynamic nature of the complex between the two proteins. We suggest that the ability to quantitate total and free IL-4 in combination with sIL-4R may provide a new insight of the role that IL-4 plays in different pathophysiological conditions.

Antibodies, Monoclonal↗
PubMed2003

Quantification of o,o'-dityrosine, o-nitrotyrosine, and o-tyrosine in cat urine samples by LC/ electrospray ionization-MS/MS using isotope dilution.

Quantification of o-tyrosine, o-nitrotyrosine, and o,o'dityrosine from cat urine samples was achieved by LC/ electrospray ionization-MS/MS (LC/ESI-MS/MS) using an isotope dilution technique in multiple reaction monitoring mode before butylation of o,o'-dityrosine and after butylation of o-tyrosine and o-nitrotyrosine. This novel approach of amino acids butylation enhanced the MS response by a factor of 7-fold for o-tyrosine and 6-fold for o-nitrotyrosine and decreased the overall chemical background noise. Butylation of o,o'-dityrosine resulted in a lower MS response as a result of the formation of both mono- and doubly butylated species. The mean recovery for the oxidized amino acids was estimated at 73 +/- 2%. The limits of quantitation of NO2-Tyr butyl ester, o-Tyr butyl ester, and di-Tyr in cat urine samples were calculated at 14.5, 28.2 and 140.9 nM, respectively. The oxidized amino acids levels in cat urine extracts ranged from 157 to 250 ng/day for o-Tyr and from 3,289 to 11,803 ng/day for di-Tyr. NO2-Tyr was found in only two urine extracts at levels below 58 ng/day. A certain trend of correlation was observed between o,o'-dityrosine and o-tyrosine when comparing these values against their respective creatinine amounts. A comparison of the data gathered from the ThermoFinnigan TSQ 7000 and Micromass Q-TOF instruments revealed several advantages of using the Q-TOF regarding the exact mass measurement, a lower ion suppression effect and the possibility to perform analyses in full scan product ion mode. These results demonstrate that a Q-TOF instrument can be a good alternative to classical triple quadrupole for quantitative purposes on a relatively small linear dynamic range (4 orders of magnitude for the Q-TOF, as compared to 6 for the triple quadrupole).

Animals↗
PubMed1999

Role of posttranscriptional regulation in circadian clocks: lessons from Drosophila.

Incredible progress has been made in the last few years in our understanding of the molecular mechanisms underlying circadian clocks. Many of the recent insights have been gained by the isolation and characterization of novel clock mutants and their associated gene products. As might be expected based on theoretical considerations and earlier studies that indicated the importance of temporally regulated macromolecular synthesis for the manifestation of overt rhythms, daily oscillations in the levels of "clock" RNAs and proteins are a pervasive feature of these timekeeping devices. How are these molecular rhythms generated and synchronized? Recent evidence accumulated from a wide variety of model organisms, ranging from bacteria to mammals, points toward an emerging trend; at the "heart" of circadian oscillators lies a cell autonomous transcriptional feedback loop that is composed of alternatively functioning positive and negative elements. Nonetheless, it is also clear that to bring this transcriptional feedback loop to "life" requires important contributions from posttranscriptional regulatory schemes. For one thing, there must be times in the day when the activities of negative-feedback regulators are separated from the activities of the positive regulators they act on, or else the oscillatory potential of the system will be dissipated, resulting in a collection of molecules at steady state. This review mainly summarizes the role of posttranscriptional regulation in the Drosophila melanogaster time-keeping mechanism. Accumulating evidence from Drosophila and other systems suggests that posttranscriptional regulatory mechanisms increase the dynamic range of circadian transcriptional feedback loops, overlaying them with appropriately timed biochemical constraints that not only engender these loops with precise daily periods of about 24 h, but also with the ability to integrate and respond rapidly to multiple environmental cues such that their phases are aligned optimally to the prevailing external conditions.

Animals↗
PubMed1999

Tolerable hearing aid delays. I. Estimation of limits imposed by the auditory path alone using simulated hearing losses.

OBJECTIVE: When people who wear hearing aids speak, there are three paths by which they hear their own voices: 1) through the air and leakage around the earmold; 2) via the solid structures of their head; 3) through the air to the hearing aid microphone, and then through the aid circuitry. These paths involve different time delays. Digital processing introduces delays in path 3 from a few to several tens of milliseconds, which could lead to a range of disturbing effects. We examined one purely auditory effect, namely hearing speech through all three of these paths. Subjective disturbance was measured as a function of delay in path 3 using simulations of hearing loss and a simulated hearing aid. With increasing hearing loss, the loudness of sound heard via paths 1 and 2 decreases, and the aid user relies more on path 3. The disturbance produced by the delay then might be less perceptible. To test this idea, four different hearing losses were simulated, varying from mild to moderately severe. DESIGN: Each of two talkers was fitted with a closed earmold, and simultaneous above-ear and in-ear recordings were made of each talker reading prose. The above-ear signal was amplified using a simulated hearing aid with 4-channel full dynamic range compression; compression ratios and gains were selected using an algorithm based on the absolute thresholds used in the simulations of hearing loss. The resultant output was then mixed with the in-ear signal with one of five values of delay, and the combined signal was processed using the four simulations of hearing loss. The resulting stimuli simulated for normal-hearing listeners the experience of having a hearing impairment and listening through a hearing aid while talking, except that the talker's voice was not that of the listener. Twenty normally hearing subjects gave subjective ratings of the disturbance of the echo for each delay and each simulated hearing loss. RESULTS: Disturbance ratings generally increased monotonically with increasing delay. Average results show that delays are rated as "disturbing" for values between 20 and 30 msec for mild to moderate losses. For a moderately severe loss, the rating "disturbing" was not quite achieved at 40 msec. For moderate losses, a speaker with low fundamental frequency (f0)(70 to 140 Hz) was less disturbing than a speaker with a medium f0, (100 to 180 Hz). This effect reversed for the mildest loss for low values of delay. CONCLUSIONS: The auditory effects of delays between bone-conducted sound and aid-conducted sound are likely to become disturbing for delays exceeding 20 msec. Somewhat longer delays may be tolerable for moderate to severe hearing losses. These delays are smaller than the delays at which audio-visual integration is disrupted.

Adult↗
PubMed1983

Dynamics of mononuclear phagocyte system Fc receptor function in systemic lupus erythematosus. Relation to disease activity and circulating immune complexes.

Seventeen pairs of longitudinal studies of mononuclear phagocyte system (MPS) Fc receptor function in 15 patients with systemic lupus were performed to explore the dynamic range of Fc receptor dysfunction in lupus and to establish the relationships between MPS function, clinical disease activity and circulating immune complexes (CIC). Fc receptor function was measured by the clearance of IgG sensitized autologous erythrocytes. At the time of first study the degree of MPS dysfunction was correlated with both clinical activity (P less than 0.05) and CIC (P less than 0.05). At follow-up patients with a change in clinical status show significantly larger changes in clearance function compared to clinically stable patients (206 min vs 7 min; P less than 0.001). MPS function changed concordantly with a change in clinical status in all cases (P = 0.002). Longitudinal assessments did not demonstrate concordance of changes in MPS function and CIC, measured by three different assays. The MPS Fc receptor defect in systemic lupus is dynamic and closely associated with disease activity. The lack of concordance of the defect with changes in CIC suggests that either CIC does not adequately reflect receptor site saturation or that other factors may also contribute to the magnitude of MPS dysfunction.

Adult↗
PubMedDate not supplied

Mammographic scanning equalization radiography.

It is well recognized that variations in breast thickness and parenchymal composition can produce a range of exposure which exceeds the latitude of high contrast mammographic film/screen combinations. Optimal imaging of the dense breast is desired since 30%-60% of women present with dense breasts, and they are believed to be at the highest relative risk of developing breast cancer. The application of scanning equalization radiography to mammography has been investigated through the construction and characterization of a prototype mammographic scanning equalization radiography (MSER) system, designed to image mammographic phantoms. The MSER system exposes a Min-R/MRH cassette by raster scanning a 2.0 x 1.6 cm beam of pulsed x-rays across the cassette. A scanning detector behind the cassette measures the local x-ray transmission of the breast. Feedback of the transmission information is used to modulate the duration of each x-ray pulse, to equalize the film exposure. The effective dynamic range of the MSER system is 25 times greater than that of conventional mammography. Artifact-free images of mammographic phantoms show that MSER effectively overcomes the latitude limitations of film/screen mammography, enabling high contrast imaging over a wide range of object x-ray transmission. Anthropomorphic phantom images show that MSER offers up to a sixfold increase in film contrast in the normally underexposed regions of conventional mammograms. Characterization of the entrance exposure shows that there is not a significant difference in exposure between MSER and conventional mammographic techniques, suggesting that both would pose comparable risk to the patient. Calculations show that the construction of a clinical multiple beam MSER system is feasible with minor changes to existing technology.

Adipose Tissue↗
PubMed2004

Measuring the nonlinear elastic properties of tissue-like phantoms.

A direct mechanical system simultaneously measuring external force and deformation of samples over a wide dynamic range is used to obtain force-displacement curves of tissue-like phantoms under plain strain deformation. These measurements, covering a wide deformation range, then are used to characterize the nonlinear elastic properties of the phantom materials. The model assumes incompressible media, in which several strain energy potentials are considered. Finite-element analysis is used to evaluate the performance of this material characterization procedure. The procedures developed allow calibration of nonlinear elastic phantoms for elasticity imaging experiments and finite-element simulations.

Adult↗
PubMed1986

Myocardial relaxation and passive diastolic properties in man.

We have developed a model for assessing the influence of the decaying contractile systolic tension on diastolic wall dynamics and the passive properties of left ventricular muscle. Total measured left ventricular diastolic pressure and stress (sigma T) are determined by two overlapping processes: the decay of actively developed pressure and stress (sigma A) and the buildup of passive filling pressure and stress (sigma*). The decaying contractile stress sigma A is formulated in terms of a relaxation pressure with a time constant (T) assessed during the isovolumic relaxation interval. By subtracting the contribution of sigma A from sigma T we obtain sigma*. With micromanometry, echocardiography, and cineangiography, total and passive stress-strain relations and strain rates were evaluated over the entire filling period in six normal control subjects and in seven patients with aortic stenosis. Elastic stiffness constants (k), the slopes of the linear passive stiffness vs sigma* relations, did not differ in the two groups over a common lower stress range (6/6 normal, k = 9.37 +/- 1.23; 7/7 aortic stenosis, k = 9.34 +/- 1.08). Over a higher sigma* range, transition into a much steeper linear region occurred, and k values were much larger (4/7 aortic stenosis, k = 28.76 +/- 2.02). When diastolic stress levels are elevated, passive stiffness-stress relations can be better described as bilinear, with a much greater wall stiffness constant in the higher than in the lower stress range. Dynamic effects of decaying systolic contractile wall stress components are important in the rapid filling phase in normal hearts as well as in those with aortic stenosis.

Angiocardiography↗
PubMed2002

An ionic liquid coating for determination of sildenafil and UK-103,320 in human serum by capillary zone electrophoresis-ion trap mass spectrometry.

Ionic liquid (IL) was covalently bonded onto the silica capillary surface and the electroosmotic flow was reversed over a pH range of 3.5 to 7. Sildenafil (SL) and its metabolite UK-103,320 (UK) in human serum were detected by solid-phase extraction followed by capillary zone electrophoresis-mass spectrometry analysis. The running buffer contained 10 mM acetic acid adjusted to pH 4.5 with 1 M ammonia, and the separating voltage was set to -25 kV. The adsorption of the analytes onto the bare capillary wall was eliminated by the IL coating and the drugs were baseline-separated within 14 min with detection limits (S/N = 3) of 14 and 17 ng/mL for SL and UK, respectively. The method developed showed good intraday precision in terms of relative standard deviation (RSD) with respect to migration time (RSD </= 0.76%, n = 5) and peak area (RSD </= 3.4%, n = 5); the relative coefficients of linear regression for both targets were better than 0.99 within linear dynamic range (SL: 50-600 ng/mL; UK: 40-400 ng/mL). Satisfactory interday precision was obtained with respect to the slope of the regression (RSD </= 4.21%, n = 9) and the migration time (RSD </= 1.1%, n = 9). The IL coating could be used with stable electroosmotic flow (RSD = 2.78%, n = 10) for at least 80 h.

Alkylation↗
PubMed2004

Factors that determine Ca2+ sensitivity of photoreceptor guanylyl cyclase. Kinetic analysis of the interaction between the Ca2+-bound and the Ca2+-free guanylyl cyclase activating proteins (GCAPs) and recombinant photoreceptor guanylyl cyclase 1 (RetGC-1).

We explored the possibility that, in the regulation of an effector enzyme by a Ca(2+)-sensor protein, the actual Ca(2+) sensitivity of the effector enzyme can be determined not only by the affinity of the Ca(2+)-sensor protein for Ca(2+) but also by the relative affinities of its Ca(2+)-bound versus Ca(2+)-free form for the effector enzyme. As a model, we used Ca(2+)-sensitive activation of photoreceptor guanylyl cyclase (RetGC-1) by guanylyl cyclase activating proteins (GCAPs). A substitution Arg(838)Ser in RetGC-1 found in human patients with cone-rod dystrophy is known to shift the Ca(2+) sensitivity of RetGC-1 regulation by GCAP-1 to a higher Ca(2+) range. We find that at physiological concentrations of Mg(2+) this mutation increases the free Ca(2+) concentration required for half-maximal inhibition of the cyclase from 0.27 to 0.61 microM. Similar to rod outer segment cyclase, Ca(2+) sensitivity of recombinant RetGC-1 is strongly affected by Mg(2+), but the shift in Ca(2+) sensitivity for the R838S mutant relative to the wild type is Mg(2+)-independent. We determined the apparent affinity of the wild-type and the mutant RetGC-1 for both Ca(2+)-bound and Ca(2+)-free GCAP-1 and found that the net shift in Ca(2+) sensitivity of the R838S RetGC-1 observed in vitro can arise predominantly from the change in the affinity of the mutant cyclase for the Ca(2+)-free versus Ca(2+)-loaded GCAP-1. Our findings confirm that the dynamic range for RetGC regulation by Ca(2+)/GCAP is determined by both the affinity of GCAP for Ca(2+) and relative affinities of the effector enzyme for the Ca(2+)-free versus Ca(2+)-loaded GCAP.

Amino Acid Substitution↗