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PubMed1991

In vivo evaluation of the semi-simultaneous method for bioavailability estimation using controlled intravenous infusion as an 'extravascular' route of administration.

A recently proposed method for bioavailability estimation, called the semi-simultaneous method, was evaluated in vivo in rats using methysergide as a test substance. In this method the test and the reference dose are administered with a short time interval and a model including the bioavailability parameters is fitted to the concentration-time profile. In the present study, in order to control the true bioavailability, intravenous infusion was used to mimic extravascular administration and various input profiles were produced. Mono-, bi- and triexponential disposition functions with the true and also various erroneous input models were fitted to the individual data sets. The models were also fitted to truncated data sets to mimic a situation where a long duration of sampling is precluded. A combined fitting-deconvolution procedure was also applied. The simi-simultaneous method gave precise and accurate estimates of the bioavailability in most groups and a robustness in the estimate concerning the model fitted was noted. The true input model could be identified for all data sets using common goodness-of-fit criteria. In the groups where a 'flip-flop' situation was created (slower input than elimination) a poorer precision and accuracy and a higher sensitivity concerning the model fitted was observed. The model fitting and the fitting-deconvolution procedure generally gave very similar results.

Animals↗
PubMed2004

In vitro dissolution and in vivo oral absorption of methylphenidate from a bimodal release formulation in healthy volunteers.

PURPOSE: The objective of this study was to evaluate the in vitro dissolution and in vivo absorption of D,L-threo-methylphenidate (MPH) from a novel bimodal release formulation (Ritalin LA capsule) compared with an immediate-release formulation (Ritalin IR tablet) in healthy volunteers. METHODS: The bimodal release formulation contains 50% of the dose in the immediate-release (IR) beads and 50% in polymethacrylate-coated, delayed-release (DR) beads. To better understand the impact of dissolution from the DR beads on oral absorption of MPH, three Ritalin LA formulations with different dissolution profiles for the DR beads (referred to as slow-, medium and fast-release formulations) were prepared, and tested together with the immediate-release formulation in 18 healthy male and female volunteers after a single oral dose under fasted conditions. The rate and extent of oral absorption of MPH were evaluated based on the overall Cmax, tmax and AUC values, as well as the Cmax, tmax and AUC values for each individual peak of the bimodal plasma concentration-time profile. The in vivo absorption-time profile was also examined by deconvolution. RESULTS: All three Ritalin LA formulations demonstrated similar bimodal plasma concentration-time profiles with two peak concentrations observed at approximately 2 and approximately 6 h post dose, mimicking that of Ritalin IR tablets given 4 h apart. Deconvolution results showed that the absorption of MPH was biphasic, with a rapid absorption phase between 0 to approximately 2 h, and a somewhat slower second absorption between approximately 3-6 h, consistent with the in vitro bimodal release characteristics of Ritalin LA formulation. The three Ritalin LA formulations were bioequivalent to one another based on the overall Cmax and AUC values and the corresponding values describing the first and second peaks, although their in vitro dissolution profiles for the DR beads were different. Compared with Ritalin IR, the Ritalin LA formulation demonstrated a similar rate of absorption for the first peak, a lower second Cmax and a higher trough concentration between peaks, as well as similar overall plasma AUC. CONCLUSIONS: Following a single oral drug administration, Ritalin LA demonstrated a two-peak plasma concentration-time profile, similar to that of the IR formulation given 4 h apart, but with less fluctuation in the plasma concentration-time profile. The in vivo biphasic absorption of MPH appeared to be well correlated with the bimodal dissolution characteristics of this new Ritalin LA formulation, and some changes in the dissolution profiles for the DR beads appeared not to affect the overall bioavailability of MPH in humans.

Administration, Oral↗
PubMed1989

Differential scanning calorimetric study of the thermal unfolding of myosin rod, light meromyosin, and subfragment 2.

The thermal unfolding of myosin rod, light meromyosin (LMM), and myosin subfragment 2 (S-2) was studied by differential scanning calorimetry (DSC) over the pH range of 6.5-9.0 in 0.5M KCl and either 0.20 M sodium phosphate or 0.15M sodium pyrophosphate. Two rod samples were examined: one was purified by Sephadex G-200 without prior denaturation (native rod), and the other was purified by a cycle of denaturation-renaturation followed by Sephacryl S-200 chromatography (renatured rod). There were clearly distinguishable differences in the calorimetric behavior of these two samples. At pH 7.0 in phosphate the DSC curves of native rod were deconvoluted into six endothermic two-state transitions with melting temperatures in the range of 46-67 degrees C and a total enthalpy of 4346 kJ/mol. Under identical conditions the melting profile of LMM was resolved into five endothermic peaks with transition temperatures in the range of 45-66 degrees C, and the thermal profile of long S-2 was resolved into two endotherms, 46 and 57 degrees C. Transition 4 observed with native rod was present in the deconvoluted DSC curve for long S-2, but absent in the DSC curve for LMM. This transition was identified with the high-temperature transition detected with long S-2 and attributed to the melting of the coiled-coil alpha-helical segment of subfragment 2 (short S-2). The low-temperature transition of long S-2 was attributed to the unfolding of the hinge region. The smallest transition temperatures observed for all three fragments were 45-46 degrees C. It is suggested that the most unstable domain in rod (domain 1) responsible for the 46 degrees C transition includes both the hinge region, which is the C-terminal segment of long S-2, and a short N-terminal segment of LMM. This domain, accounting for 21% of the rod structure, contains the S-2/LMM junction, and upon proteolytic cleavage yields the C-terminal and N-terminal ends of long S-2 and LMM, respectively. Over the pH range of 6.5-7.5, the observed specific heat of denaturation of rod was approximately equal to the sum of the specific heats of LMM and S-2. This finding provides an additional argument for the existence of independent domains in myosin rod.

Animals↗
PubMed2006

Functional phenotyping of human plasma using a 361-fluorogenic substrate biosensing microarray.

A microarray presenting glycerol nanodroplets of fluorogenic peptide substrates was used as a biosensor for the detection of multiple enzyme activities within human plasma. Using 10 different plasma proteases (kallikrein, factor XIIa, factor XIa, factor IXa, factor VIIa, factor Xa, thrombin, activated protein C, uPA and plasmin) and a 361-compound fluorogenic substrate library (Ac-Ala-P3-P2-Arg-coumarin for P = all amino acids except Cys), a database was created for deconvoluting the relative activity of each individual enzyme signal in human plasma treated with various activators (calcium, kaolin, or uPA). Three separate deconvolution protocols were tested: searching for "optimal" sensing substrate sequences for a set of 5 enzymes and using these substrates to detect protease signals in plasma; ranking the "optimal" sensing substrates for 10 proteases using local error minimization, resulting in a set of substrates which were bundled via weighted averaging into a super-pixel that had biosensing properties not obtainable by any individual fluorogenic substrate; and treating each 361-element map measured for each plasma preparation as a weighted sum of the 10 maps obtained for the 10 purified enzymes using a global error minimization. The similarity of the results from these latter two protocols indicated that a small subset of <90 substrates contained the majority of biochemical information. The results were consistent with the state of the coagulation cascade expected when treated with the given activators. This method may allow development of future biosensors using minimal and non-specific markers. These substrates can be applied to real-time diagnostic biosensing of complex protease mixtures.

Algorithms↗
PubMed1990

Kinetics of intestinal calcium absorption in humans measured using stable isotopes and high-precision thermal ionization mass spectrometry.

Oral (44Ca: 0.13-0.20 mmol) and intravenous (42Ca: 0.02-0.037 mmol) isotopically enriched stable calcium (Ca) tracers were given together with an oral dose of 2.5 mmol of natural Ca to normal subjects. Blood and urine samples were collected up to 24 h after the tracer doses and atom fractions (AFs) of these tracers (relative to natural Ca) were measured by high-precision thermal ionization mass spectrometry (TIMS). The time-dependent fractional rate of oral dose absorbed and true fractional intestinal Ca absorption (alpha) were derived from the Afs by mathematical deconvolution. After 6 h, the ratio AF oral tracer/AF intravenous tracer in blood equalled that in urine and did not change thereafter. Reproducibility of the combination of chemical precipitation of Ca (from a urine standard) and subsequent TIMS measurements, in nine runs over 13 months, was 1.2% (coefficient of variation). This was in accord with the within-run reproducibility. An estimate of alpha derived from a single blood or urine measurement was 6-10% higher than the reference value obtained by deconvolution. This discrepancy could be explained by a correction factor depending, in part, on the elapsed time for peak Ca intestinal absorption rate. Instrumentally induced mass fractionation, as well as contributions from radiogenic Ca, had a significant effect on the accuracy and reproducibility of the ratio of AFs of tracers in blood and urine.

Administration, Oral↗
PubMed1983

Long-term storage of samples for flow cytometric DNA analysis.

A simple procedure for long-term storage of cells for flow cytometric DNA analysis was developed and tested. The cells were stored as single cells or fine-needle aspirates suspended in a citrate buffer with dimethylsulfoxide (DMSO), or as small blocks of tissue from solid tumors. The cells were stored for up to one year by freezing at -80 degrees C. Statistical analysis of the results showed no change in the fractions of cells in the cell cycle phases as determined by deconvolution of the DNA-histograms. It was found that in addition to the intrinsic sample variation from the parameter estimation by deconvolution, there was significant intraday and interday variation. Hence the most accurate results are obtained if different aliquots of a sample are measured on different days rather than on the same day. Use of the storage method thus has the potential of increasing the accuracy of the analysis. The storage method makes sample collection independent of immediate subsequent analysis. This has enabled us to perform large internally controlled experiments, involving more samples than can be analyzed in one day, to examine tumor samples from different hospitals and to utilize fully the capacity of our flow cytometer. The method was a prerequisite for developing an accurate standardization procedure for DNA content determination.

Analysis of Variance↗
PubMed2006

Single particle high resolution spectral analysis flow cytometry.

BACKGROUND: While conventional multiparameter flow cytometers have proven highly successful, there are several types of analytical measurements that would benefit from a more comprehensive and flexible approach to spectral analysis including, but certainly not limited to spectral deconvolution of overlapping emission spectra, fluorescence resonance energy transfer measurements, metachromic dye analysis, free versus bound dye resolution, and Raman spectroscopy. METHODS: Our system utilizes a diffraction grating to disperse the collected fluorescence and side-scattered light from cells or microspheres passing through the interrogation region over a rectangular charge-coupled-device image sensor. The flow cell and collection optics are taken from a conventional flow cytometer with minimal modifications to assure modularity of the system. RESULTS: Calibration of the prototype spectral analysis flow cytometer included wavelength characterization and calibration of the dispersive optics. Benchmarking of the system demonstrated a single particle/cell intensity sensitivity of 2160 MESF of R-Phycoerythrin. Single particle spectra taken with our instrument were validated against bulk solution fluorimeter and conventional flow cytometer measurements. Coefficients of variation of integrated spectral fluorescence intensity of several sets of standard fluorescent microspheres ranged from 1.4 to 4.8% on the spectral system. Spectral discrimination of free versus PI bound to cells is also demonstrated. CONCLUSIONS: It is demonstrated that the flow spectrometer has sufficient sensitivity and wavelength resolution to detect single cells and microspheres, including multi-fluorophore labeled microspheres. The capability to use both standard mathematical deconvolution techniques for data analysis, coupled with the feasibility of integration with existing flow cytometers, will improve the accuracy and precision of ratiometric measurements, enable the analysis of more discrete emission bands within a given wavelength range, and allow more precise resolution of the relative contribution of individual fluorophores in multiply-tagged samples, thereby enabling a range of new applications involving the spectral analysis of single cells and particles.

Calibration↗
PubMed2006

Multicolor in vivo time-lapse imaging at cellular resolution by stereomicroscopy.

Intravital time-lapse imaging has altered significantly many long-standing rules of biological mechanisms, but being apparatus-intense and laborious, time-lapse imaging remained mostly restricted to specialized labs. We show that recently introduced, fully automated fluorescence stereomicroscopes represent cost-effective but powerful means of imaging dynamic events ranging from observing embryogenesis over several days to detailed tissue rearrangements and fast blood cell rolling in vivo. When combined with deconvolution approaches, even subcellular resolution in several colors can be achieved. Using three-dimensional image recording, we show the spatial reconstruction of expression patterns. Furthermore, by combining three-dimensional image recording over time with subsequent deconvolution analysis, we demonstrate that subcellular dynamics such as axonal pathfinding can be resolved. These findings promise that time-lapse imaging using a stereomicroscope will become a hands-on standard method for phenotype analysis in many fields of biology.

Animals↗
PubMed2005

Development of a quality control method for the characterization of oligonucleotides by capillary zone electrophoresis-electrospray ionization-quadrupole time of flight-mass spectrometry.

A capillary zone electrophoresis-negative electrospray ionization-quadrupole time of flight-mass spectrometric method was developed for the characterization of oligonucleotides after synthesis, using model compounds. The major difficulty is the adduction of metal cations to the polyanionic backbone of the oligonucleotide sample, resulting in complex spectra and decreased sensitivity. Several approaches were investigated to circumvent this problem. Separation was performed in an ammonium carbonate buffer. During separation, the interfering metal ions were exchanged for ammonium ions, which are less tightly bound to the oligonucleotide when ionized. The influence of the addition of piperidine and imidazole or trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) to the running buffer for further reduction of cation adduction was investigated. Addition of CDTA to the buffer system resulted in a deconvoluted spectrum with very little adducts. On-line sample stacking proved vital to preconcentrate the samples. The pH and the concentration of the ammonium carbonate buffer as well as the electrophoresis voltage were optimized to achieve the best signal response for the oligonucleotides and a maximum reduction of the cation adducts as well as a short analysis time. Finally, the sheath liquid composition was examined for further improvement of the signal. The developed method was used to analyze different oligonucleotides (5000-9200 Da) in light of its use as a final quality control method for oligonucleotides in terms of purity and sequence homogeneity of the synthesized products. In all cases, very little adducts were observed in the deconvoluted spectra, and the relative errors of the measured molecular masses ranged from 3 to 35 ppm.

Buffers↗
PubMed1991

Long-term enhancement of CA1 synaptic transmission is due to increased quantal size, not quantal content.

Quantal components of Schaffer collateral synaptic transmission recorded intracellularly from CA1 pyramidal cells were examined using 2 methods: simultaneous recordings of CA3-CA1 cell-pairs, and minimal electrical stimulation in stratum radiatum. Quantal parameters estimated by the method of failures and by a computer algorithm that optimized parameter estimates using deconvolution of background noise were highly correlated. EPSP-amplitude histograms of CA3-CA1 cell pairs (N = 10) and minimal electrical stimulation (N = 33) could be adequately described either by Poisson or binomial statistics, or by both, and exhibited similar estimates of unit quantal size (q) and mean quantal content (m). Paired-pulse stimulation with 50 msec between stimuli resulted in an expected facilitation in the EPSP amplitude and increase in m during the second response, as estimated by noise deconvolution, by the decrease in apparent failures, and by a decrease in the coefficient of variation of the EPSP. Tetanization of the Schaffer collaterals that induced long-term enhancement (LTE/LTP) of the population response was associated with an average increase in q for minimal-stimulation responses, with no significant change in any estimate of m. Taken together, these data indicate that, under the present experimental conditions, LTE is expressed as an increase in quantal size, rather than an increase in the number of quanta released per presynaptic impulse. Although this is not definitive evidence for a postsynaptic mechanism, these findings do further restrict the classes of possible presynaptic mechanisms that may be proposed to account for LTE expression.

Action Potentials↗
PubMed1995

Pharmacokinetics, absolute bioavailability, and absorption characteristics of lamivudine.

Lamivudine is a novel cytosine nucleoside analog, reverse transcriptase inhibitor that has shown activity against human immunodeficiency virus (HIV) types 1 and 2 and hepatitis B virus in vitro. This study was conducted to compare the absolute bioavailability, pharmacokinetics, and absorption characteristics of oral solution, 100-mg capsule, and 100-mg tablet formulations of lamivudine with those of intravenous lamivudine. Twelve patients with HIV were enrolled in a single-center, randomized, open-label, four-way cross-over study. Treatment arms consisted of 100 mg intravenous lamivudine (administered over 1 hour), 100 mg oral lamivudine (1 mg/mL), a 100-mg capsule, and a 100-mg tablet, each followed by a 3- to 14-day washout period. Serial blood samples over 24 hours were obtained after each dose administration. Serum concentration data were analyzed to determine pharmacokinetic parameter estimates including area under the curve (AUC), terminal half-life (t1/2), mean residence time (MRT) for each formulation, systemic clearance, oral clearance, and apparent volume of distribution (Vd). Absolute bioavailability and in vivo mean absorption time (MAT) and mean dissolution time (MDT) were calculated for the oral formulations. Deconvolution techniques were used to calculate the input rate for the oral solution, capsule, and tablet. The two one-sided t test was used to determine bioequivalency among oral formulations with respect to logarithmic transformed estimates of AUC and maximum peak concentration (Cmax). Mean (CV) systemic clearance and Vdss after intravenous administration of lamivudine were 22.6 L/h (15%) and 99 L (28%), respectively; mean t1/2 ranged from 8.41 to 9.11 hours for all formulations; and MRT ranged from 4.42 to 5.77 hours for all formulations. Mean absolute bioavailability ranged from 86% to 88% for the oral solution, capsule, and tablet. All oral formulations were considered bioequivalent for AUC and Cmax. The MAT was 1.32 hour for the oral solution, and MDT was 0.03 and -0.11 hours for the capsule and the oral solution, respectively. The oral formulations of lamivudine examined in this study demonstrated acceptable bioavailability for oral administration. The solid oral formulations (capsule and tablet) show rapid dissolution properties with an absorption rate similar to or exceeding those observed with the oral solution. This suggests that dissolution is not an important factor for the rate of absorption of lamivudine. The use of deconvolution techniques using PCDCON provides valuable insight into the absorption characteristics of lamivudine.

Absorption↗
PubMed2003

Changes in spinal GDNF, BDNF, and NT-3 expression after transient spinal cord ischemia in the rat.

Previous studies have demonstrated that the expression of several growth factors including glial cell-derived neurotrophic factor (GDNF), brain-derived growth factor (BDNF), and neurotrophin-3 (NT-3) play an important role in defining neuronal survival after brain ischemia. In the present study, using a well-defined model of transient spinal ischemia in rat, we characterized the changes in spinal GDNF, BDNF, and NT-3 expression as defined by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence coupled with deconvolution microscopy. In control animals, baseline levels of GDNF, BDNF, and NT-3 (74 +/- 22, 3,600 +/- 270, 593 +/- 176 pg/g tissue, respectively) were measured. In the ischemic group, 6 min of spinal ischemia resulted in a biphasic response with increases in tissue GDNF and BDNF concentrations at the 2-hr and 72-hr points after ischemia. No significant differences in NT-3 concentration were detected. Deconvolution analysis revealed that the initial increase in tissue GDNF concentration corresponded to a neuronal upregulation whereas the late peak seen at 72 hr corresponded with increased astrocyte-derived GDNF synthesis. Increased expression of BDNF was seen in neurons, astrocytes, and oligodendrocytes. These data suggest that the early increase in neuronal GDNF/BDNF expression likely modulates neuronal resistance/recovery during the initial period of postischemic reflow. Increased astrocyte-derived BDNF/GDNF expression corresponds with transient activation of astrocytes and may play an active role in neuronal plasticity after non-injurious intervals of spinal ischemia.

Animals↗
PubMed2002

Intestinal absorption and presystemic elimination of the prokinetic agent, EM574, in the rabbit.

The purpose of this study was to characterize the pharmacokinetics and dose proportionality of the prokinetic macrolide, EM574, in rabbits following intravenous dosing, and to determine the intestinal absorption and intestinal and hepatic first-pass elimination of EM574 in rabbits. Two doses (0.05 and 0.25 mg/kg) of EM574 were given to rabbits intravenously in a crossover study. In a separate gut perfusion study, rabbit duodenal or jejunal segments were perfused with EM574 solution at 0.2 mL/min for 130 min. Plasma levels of EM574 were determined by a validated LC-MS/MS assay, and concentrations in perfusate were determined by HPLC with UV detection. The absorptive clearance (PeA) of EM574 was calculated from the steady-state rate of disappearance from the gut lumen during perfusion. The cumulative amount (A(app)) of drug appearing in the systemic circulation was calculated by deconvolution, where the input response was the plasma concentration-time profile during intestinal perfusion and the unit impulse response was the mean profile following intravenous bolus dosing to sham-operated rabbits in a separate experiment. F(g)F(h) was calculated from the ratio of A(app) to the total amount disappeared from gut lumen during perfusion. Hepatic first-pass elimination was measured by intraportal venous infusion. EM574 exhibits linear kinetics over the dose range studied. CL, V(ss), and terminal half-life (mean +/- SD) of EM574 were 68.6 +/- 15.5 mL/min/kg, 13.4 +/- 3.0 L/kg, and 2.7 +/- 0.8 h, respectively. EM574 is expected to be absorbed completely from the rabbit small intestine based on its high jejunal PeA values (8.1 +/- 2.2, and 5.5 +/- 1.5 microL/min/cm following low and high dose perfusion, respectively). The first-pass extraction of EM574 was substantial and dose independent. Mean F(g) and F(h) were 0.14 and 0.20, respectively, suggesting that the intestinal and hepatic first-pass elimination of EM574 were comparable. Deconvolution was successfully applied in the determination of gut wall and hepatic first-pass elimination of EM574.

Administration, Oral↗
PubMed2003

Quantification of bolus-tracking MRI: Improved characterization of the tissue residue function using Tikhonov regularization.

Quantification of cerebral blood flow (CBF) and the tissue residue function (R) using bolus-tracking MRI requires deconvolution of the arterial input function (AIF). Currently, the most commonly used deconvolution method is singular value decomposition (SVD), which has been shown to produce accurate estimations of CBF. However, this method introduces unwanted oscillations in the time course of R, and there are situations in which the actual shape is of interest (e.g., in calculating flow heterogeneity and assessing bolus dispersion). In such cases, the conventional SVD method may no longer be suitable, and an alternative approach may be required. This work describes the implementation of Tikhonov regularization with the L-curve criterion to quantify CBF and obtain a better characterization of R. The methodology is tested on simulated and patient data, and the results are compared to those found using the conventional SVD approach. Although both methods produce similar CBF values, the deconvolved R shape obtained using SVD is dominated by oscillations and fails to characterize the shape in the presence of dispersion. On the other hand, the use of the proposed regularization method improves the characterization of the tissue residue function.

Carotid Stenosis↗
PubMed1991

An evaluation of new processing protocols for in vivo NMR spectroscopy.

In vivo NMR spectroscopy is often complicated with problems of low signal-to-noise, poor resolution, undefined peak shapes, and nonlinear baselines despite the efforts of investigators to optimize their experiments. Several data processing options are available to spectroscopists to enhance resolution and signal-to-noise and/or to flatten baselines. There is some question about how these processing protocols affect quantitative information. This paper evaluates five different processing protocols for their ability to extract quantitative information from a set of nonideal spectra. Three of the protocols involve recently developed statistical signal processing methods, maximum entropy Fourier spectral deconvolution, linear prediction singular value decomposition, and baseline deconvolution. These protocols are compared with the conventional processing methods of convolution difference and zeroing initial data points of the FID. The methods are evaluated by use of a quantitative 31P model sample and also are demonstrated on surface coil 31P data.

2,3-Diphosphoglycerate↗
PubMed2005

Advantages of frequency-domain modeling in dynamic-susceptibility contrast magnetic resonance cerebral blood flow quantification.

In dynamic-susceptibility contrast magnetic resonance perfusion imaging, the cerebral blood flow (CBF) is estimated from the tissue residue function obtained through deconvolution of the contrast concentration functions. However, the reliability of CBF estimates obtained by deconvolution is sensitive to various distortions including high-frequency noise amplification. The frequency-domain Fourier transform-based and the time-domain singular-value decomposition-based (SVD) algorithms both have biases introduced into their CBF estimates when noise stability criteria are applied or when contrast recirculation is present. The recovery of the desired signal components from amid these distortions by modeling the residue function in the frequency domain is demonstrated. The basic advantages and applicability of the frequency-domain modeling concept are explored through a simple frequency-domain Lorentzian model (FDLM); with results compared to standard SVD-based approaches. The performance of the FDLM method is model dependent, well representing residue functions in the exponential family while less accurately representing other functions.

Algorithms↗
PubMed2006

Model-free arterial spin labeling quantification approach for perfusion MRI.

In this work a model-free arterial spin labeling (ASL) quantification approach for measuring cerebral blood flow (CBF) and arterial blood volume (aBV) is proposed. The method is based on the acquisition of a train of multiple images following the labeling scheme. Perfusion is obtained using deconvolution in a manner similar to that of dynamic susceptibility contrast (DSC) MRI. Local arterial input functions (AIFs) can be estimated by subtracting two perfusion-weighted images acquired with and without crusher gradients, respectively. Furthermore, by knowing the duration of the bolus of tagged arterial blood, one can estimate the aBV on a voxel-by-voxel basis. The maximum of the residue function obtained from the deconvolution of the tissue curve by the AIF is a measure of CBF after scaling by the locally estimated aBV. This method provides averaged gray matter (GM) perfusion values of 38 +/- 2 ml/min/100 g and aBV of 0.93% +/- 0.06%. The average CBF value is 10% smaller than that obtained on the same data set using the standard general kinetic model (42 +/- 2 ml/min/100 g). Monte Carlo simulations were performed to compare this new methodology with parametric fitting by the conventional model.

Algorithms↗
PubMed1996

Intrinsic tryptophans of CRABPI as probes of structure and folding.

The native state fluorescence and CD spectra of the predominantly beta-sheet cellular retinoic acid-binding protein I (CRABPI) include contributions from its three tryptophan residues and are influenced by the positions of these residues in the three-dimensional structure. Using a combination of spectroscopic approaches and single Trp-mutants of CRABPI, we have deconvoluted these spectra and uncovered several features that have aided in our analysis of the development of structure in the folding pathway of CRABPI. The emission spectrum of native CRABPI is dominated by Trp 7. Trp 109 is fluorescence-silent due to its interaction with the guanidino group of Arg 111. Although the far-UV CD spectrum of CRABPI is largely determined by the protein's secondary structure, aromatic clustering around Trp 87 and the aromatic-charge interaction between Arg 111 and Trp 109 give rise to a characteristic feature in the CD spectrum at 228 nm. The near-UV CD bands of CRABPI arise largely from additive contributions of the three tryptophan residues. Trp 7 and Trp 87 give a negative CD band at 275 nm. The near-UV CD band from Trp 109 is positive and shifted to longer wavelengths (to 302 nm) due to the charge-aromatic interaction between Arg 111 and Trp 109. Our deconvolution of the equilibrium spectra have been used to interpret kinetic folding experiments monitored by stopped-flow fluorescence. These dynamic experiments suggest the early evolution of a well-populated, hydrophobically collapsed intermediate, which undergoes global rearrangement to form the fully folded structure. The results presented here suggest several additional strategies for dissecting the folding pathway of CRABPI.

Circular Dichroism↗