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Results for “parameter optimization”

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PubMed1990

Syntheses of argininal semicarbazone containing peptides and their applications in the affinity chromatography of serine proteinases.

Eight argininal semicarbazone containing peptides prepared by liquid phase synthesis were all found to be reversible inhibitors of model serine proteinases including trypsin and plasma kallikrein (PK). Among the peptides tested, those having a Lys residue at position P2 displayed the maximum binding potency towards PK. One of the peptides, Leu-enkephalin-argininal semicarbazone, a comparatively weak inhibitor, was chosen in order to develop an affinity-based purification protocol for PK. The affinity column was prepared by covalent attachment of the NH2-terminal moiety of the peptidyl semicarbazone to a solid-phase matrix bearing a spacer group. For efficient binding of PK, it was found necessary to optimize parameters like the concentration of inhibitor linked to the solid matrix, the ionic strength of the buffer used, the temperature and the pH. The majority of the bound enzyme could be recovered following elution with guanidine hydrochloride or benzamidine hydrochloride in a high salt buffer at pH 6.0. The usefulness of the affinity procedure towards the purification of other serine proteinases is also discussed.

Amino Acid Sequence↗

PubMed1994

DNA fingerprinting of Eikenella corrodens by pulsed-field gel electrophoresis.

In order to conduct molecular typing of Eikenella corrodens strains by macrorestriction fingerprinting, we evaluated different restriction enzymes for digestion of genomic DNA and determined the optimal parameters for separating E. corrodens DNA by pulsed-field gel electrophoresis. Ten E. corrodens strains isolated from oral and extraoral infection sites in different individuals were analyzed. The rare-cutting restriction endonucleases DraI, SmaI and XbaI usually used for pulsed-field gel electrophoresis analyses were not suitable for digestion of E. corrodens genomic DNA because they either did not digest the DNA or produced bands of similar molecular weights that could not be separated. Accordingly, among additional enzymes including BamHI, BglII, EcoRI and Hind III, we found BamHI and BglII to be the most suitable rare-cutting enzymes for pulsed-field gel electrophoresis analysis. They cleaved the genomes of all the above strains into 15-20 fragment bands that were clearly separated by the following pulsed-field gel electrophoresis conditions: 140 V with a running time of 40 h, pulse times of 5 to 50 s with linear ramping and an electrical field angle of 120 degrees. These conditions enabled us to distinguish 8 individual pulsed-field gel electrophoresis patterns from the 10 strains analyzed. However, only 4 identical outer membrane protein profiles were differentiated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data obtained in this analysis showed clonal divergence among members of the E. corrodens species, at the same time revealing this pulsed-field gel electrophoresis as being a highly attractive procedure for epidemiological investigation of this organism, including its acquisition and transmission.

Bacterial Outer Membrane Proteins↗

PubMed1994

An antibody screening test based on the antiglobulin gel technique, pooled test cells, and plasma.

The recently introduced gel technique offers significantly advances compared to traditional tube tests. The purpose of the present study was to design an antibody screening test based on the gel technique, pooled cells, and plasma and to evaluate this test by comparison with a conventional spin-tube indirect antiglobulin test (IAT) combined with a two-stage papain technique. Pilot studies were performed to establish optimal parameters during the design phase, and finally 5,446 consecutive samples were screened for irregular antibodies by the gel technique in parallel with routine techniques. Irregular erythrocyte antibodies were detected in 151 samples, and the gel technique proved equal or superior to the tube test in the detection of all antibodies except 'enzyme-only' anti-E and anti-Lea. We conclude from this study that screening for unexpected antibodies using the gel IAT in our set-up, which includes: (1) omission of enzyme technique; (2) the use of stabilized (EDTA) plasma instead of serum as test material in order to facilitate automation; and (3) pooling 2 by 2 of 4 test cells (to make the gel technique price competitive), is a fast, reliable and sensitive procedure that maintains transfusion safety and compares favourably with our previous routine of saline IAT combined with a two-stage papain technique.

ABO Blood-Group System↗

PubMed1995

An improved Escherichia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp. using electroporation.

The genetic studies of metabolically diverse Rhodococcus spp. have been hampered by the lack of a system of introducing exogenous DNA. The authors improved an existing Escherichia coli-Rhodococcus shuttle vector (pMVS301) by removing much of the DNA not needed for replication and adding a multicloning site. This improved vector (pBS305) is 7.9 kb in length. Its ability to transform Rhodococcus was tested using electroporation parameters optimized for introduction of pMVS301 into Rhodococcus. Transformation efficiencies as high as 10(5) cfu micrograms-1 DNA were obtained although efficiencies varied depending on the Rhodococcus strain tested. The improved vector pBS305 offers great utility for genetic studies of Rhodococcus because its small size enables movement of large inserts of DNA into Rhodococcus, it has multicloning sites, contains a highly selective thiostrepton marker, and can be replicated in both E. coli and Rhodococcus.

DNA, Bacterial↗

PubMed1994

Non-adrenergic non-cholinergic (NANC) excitatory response of the channel catfish intestine.

1. Optimal parameters for electrical field stimulation (EFS) of catfish pyloric and middle intestinal segments were determined (15 Hz, 60 V) from a range of frequencies (5-45 Hz) and voltages (40-120 V) using a modified Magnus' method. Contractile responses were produced by EFS which were reproducible and showed no significant difference between the tissues. 2. The contractile cholinergic responses of the tissues to carbachol and acetylcholine (ACh) were blocked by atropine on an equimolar concentration, whereas, these responses were enhanced in the presence of neostigmine, and acetylcholinesterase inhibitor. 3. Adrenergic responses were examined with noradrenaline (NA). NA produced contraction of the segments only, at a concentration of 10(-4) M. Among the various adrenoceptors, beta-adrenoceptor stimulation produced a weak relaxation whereas, both alpha 1- and alpha 2-adrenoceptor stimulation produced contractions, of which alpha 2-induced contraction was of greater magnitude. The beta, alpha 1 and alpha 2 responses were blocked by their respective blocking agents propranolol, prazosin and yohimbine. 4. The autonomic components of the response to EFS were determined by using selected cholinergic and adrenergic antagonists separately or collectively. Cholinergic blockade with atropine did not produce a significant blockade of the EFS-induced response. Similarly, blockade of beta-adrenoceptors with propranolol did not modulate the contractile response to EFS to any significant level. Blockade by prazosin or yohimbine did not significantly change the contractile response to EFS. After a complete blockade of the adrenergic and cholinergic divisions, the intestinal segments still showed a contractile response to EFS which was not significantly different from the control response. This indicated the presence of a non-adrenergic non-cholinergic (NANC) response. 5. Tetrodotoxin, at 10(-6) M, significantly blocked the EFS-induced NANC response suggesting a neurogenic origin for the response. 6. The present study indicated that the EFS-induced response of the catfish intestinal segments is predominantly NANC-e in nature suggesting an important role for it in the regulation of intestinal motility.

Acetylcholine↗

PubMed1986

A new technique for laser treatment of cutaneous tumors.

The CO2 laser was used to treat three patients with discrete, cutaneous tumors. For each patient, we established the relationship between the width of the lesions and their maximum thickness, and between pulse energy and the depth of the resultant crater. Optimal parameters for therapy were established from these results.

Adolescent↗

PubMed2006

Early ethanol's anxiolytic effects assessed through an unconditional stimulus revaluation procedure.

BACKGROUND: Even though ethanol is considered to exert antianxiety effects, the existence of such properties during early ontogeny of the rat has rarely received attention, mainly because of the restricted behavioral repertoire of the pup. This study evaluated ethanol's anxiolytic properties in preweanling rats through an unconditional stimulus revaluation procedure. METHODS: Optimal parameters for the acquisition of an odor avoidance response were obtained in Experiment 1. In Experiment 2 preweanlings experienced 4 conditioning trials defined by a 5 minute exposure to a lemon odor while they were intraorally infused with a sapid aversive stimulus (citric acid). Twenty-four hours later, pups were briefly exposed to the acid 5 minutes after being administered with 0.0, 0.25, 0.5, 1.25, or 2.5 g/kg of ethanol. Odor preferences were then evaluated in a 2-way odor preference test. Blood ethanol concentrations (BEC's) derived from these doses were assessed in Experiment 3. In Experiment 4, citric acid mediated-conditioning was followed by a similar odor-preference test after pups were re-exposed to the acid either 5 or 25 minutes after receiving a 2.5 g/kg ethanol dose. Ethanol's effects upon the avoidance response were also analyzed in an alternative rat strain (Experiment 5). RESULTS: Moderate to low ethanol doses ameliorated the otherwise strong avoidance response. Further experiments: (a) replicated these phenomena, (b) established that they are correlated with BEC's in the range of 17-70 mg%, and (c) Indicated that ethanol's influence upon an avoidance response changes as a function of the temporal course of the intoxication. CONCLUSIONS: Acute ethanol interacts with aversive nondrug-mediated learning. These findings are likely to be discussed in terms of possible negative reinforcing properties of ethanol and indicate that this drug exerts differential motivational effects during the course of the intoxication. The devaluation procedure should be considered as an alternative method to assess ethanol's motivational properties.

Alcoholic Intoxication↗

PubMed2005

Stratified analysis in randomized trials with noncompliance.

This article develops methods for stratified analyses of additive or multiplicative causal effect on binary outcomes in randomized trials with noncompliance. The methods are based on a weighted estimating function for an unbiased estimating function under randomization in each stratum. When known weights are used, the derived estimator is a natural extension of the instrumental variable estimator for stratified analyses, and test-based confidence limits are solutions of a quadratic equation in the causal parameter. Optimal weights that maximize asymptotic efficiency incorporate variability in compliance aspects across strata. An assessment based on asymptotic relative efficiency shows that a substantial enhancement in efficiency can be gained by using optimal weights instead of conventional ones, which do not incorporate the variability in compliance aspects across strata. Application to a field trial for coronary heart disease is provided.

Cholesterol↗

PubMedDate not supplied

Introducing antisense oligodeoxynucleotides into Paramecium via electroporation.

A method utilizing electroporation to deliver antisense oligodeoxynucleotides into Paramecium tetraurelia has been developed. For these studies antisense oligonucleotides directed to different regions of the calmodulin mRNA were used. It was found that a pulse delivered at 150-250 V (375-625 V/cm field strength) for 3.9-4.2 ms using a 275 microF capacitor with resistance set at 13 Ohms was sufficient to achieve measurable incorporation of fluorescently-labeled oligodeoxynucleotides in up to 95% of the cells treated. Optimal parameters included using oligodeoxynucleotides of at least 12 bases in length with a 3' blocking group at a dose of around 10 microM. In addition, multiple oligodeoxynucleotides directed to the same target mRNA resulted in at least a 10-fold reduction in the dose of oligodeoxynucleotide required to achieve the desired effects. Taken together, these results indicate that the use of antisense oligodeoxynucleotides can be an easy and useful method for linking genes to specific functions in Paramecium tetraurelia. Finally, this report discusses how different 3' blocking groups and the use of combinations of oligodeoxynucleotides directed to different regions of the same target mRNA can help address concerns about specificity.

Animals↗

PubMed2006

A simple in vivo assay for measuring the efficiency of gene length-dependent processes in yeast mRNA biogenesis.

We have developed a simple reporter assay useful for detection and analysis of mutations and agents influencing mRNA biogenesis in a gene length-dependent manner. We have shown that two transcription units sharing the same promoter, terminator and open reading frame, but differing in the length of their 3'-untranslated regions, are differentially influenced by mutations affecting factors that play a role in transcription elongation or RNA processing all along the transcription units. In contrast, those mutations impairing the initial steps of transcription, but not affecting later steps of mRNA biogenesis, influence equally the expression of the reporters, independently of the length of their 3'-untranslated regions. The ratio between the product levels of the two transcription units is an optimal parameter with which to estimate the efficiency of gene length-dependent processes in mRNA biogenesis. The presence of a phosphatase-encoding open reading frame in the two transcription units makes it very easy to calculate this ratio in any mutant or physiological condition. Interestingly, using this assay, we have shown that mutations in components of the SAGA complex affect the level of mRNA in a transcript length-dependent fashion, suggesting a role for SAGA in transcription elongation. The use of this assay allows the identification and/or characterization of new mutants and drugs affecting transcription elongation and other related processes.

Acid Phosphatase↗

PubMed2006

Reduction of a biochemical model with preservation of its basic dynamic properties.

The complexity of full-scale metabolic models is a major obstacle for their effective use in computational systems biology. The aim of model reduction is to circumvent this problem by eliminating parts of a model that are unimportant for the properties of interest. The choice of reduction method is influenced both by the type of model complexity and by the objective of the reduction; therefore, no single method is superior in all cases. In this study we present a comparative study of two different methods applied to a 20D model of yeast glycolytic oscillations. Our objective is to obtain biochemically meaningful reduced models, which reproduce the dynamic properties of the 20D model. The first method uses lumping and subsequent constrained parameter optimization. The second method is a novel approach that eliminates variables not essential for the dynamics. The applications of the two methods result in models of eight (lumping), six (elimination) and three (lumping followed by elimination) dimensions. All models have similar dynamic properties and pin-point the same interactions as being crucial for generation of the oscillations. The advantage of the novel method is that it is algorithmic, and does not require input in the form of biochemical knowledge. The lumping approach, however, is better at preserving biochemical properties, as we show through extensive analyses of the models.

Algorithms↗

PubMedDate not supplied

Regional estimation of base recharge to ground water using water balance and a base-flow index.

Naturally occurring long-term mean annual base recharge to ground water in Nebraska was estimated with the help of a water-balance approach and an objective automated technique for base-flow separation involving minimal parameter-optimization requirements. Base recharge is equal to total recharge minus the amount of evapotranspiration coming directly from ground water. The estimation of evapotranspiration in the water-balance equation avoids the need to specify a contributing drainage area for ground water, which in certain cases may be considerably different from the drainage area for surface runoff. Evapotranspiration was calculated by the WREVAP model at the Solar and Meteorological Surface Observation Network (SAMSON) sites. Long-term mean annual base recharge was derived by determining the product of estimated long-term mean annual runoff (the difference between precipitation and evapotranspiration) and the base-flow index (BFI). The BFI was calculated from discharge data obtained from the U.S. Geological Survey's gauging stations in Nebraska. Mapping was achieved by using geographic information systems (GIS) and geostatistics. This approach is best suited for regional-scale applications. It does not require complex hydrogeologic modeling nor detailed knowledge of soil characteristics, vegetation cover, or land-use practices. Long-term mean annual base recharge rates in excess of 110 mm/year resulted in the extreme eastern part of Nebraska. The western portion of the state expressed rates of only 15 to 20 mm annually, while the Sandhills region of north-central Nebraska was estimated to receive twice as much base recharge (40 to 50 mm/year) as areas south of it.

Environmental Monitoring↗

PubMedDate not supplied

Effects of model sensitivity and nonlinearity on nonlinear regression of ground water flow.

Nonlinear regression is increasingly applied to the calibration of hydrologic models through the use of perturbation methods to compute the Jacobian or sensitivity matrix required by the Gauss-Newton optimization method. Sensitivities obtained by perturbation methods can be less accurate than those obtained by direct differentiation, however, and concern has arisen that the optimal parameter values and the associated parameter covariance matrix computed by perturbation could also be less accurate. Sensitivities computed by both perturbation and direct differentiation were applied in nonlinear regression calibration of seven ground water flow models. The two methods gave virtually identical optimum parameter values and covariances for the three models that were relatively linear and two of the models that were relatively nonlinear, but gave widely differing results for two other nonlinear models. The perturbation method performed better than direct differentiation in some regressions with the nonlinear models, apparently because approximate sensitivities computed for an interval yielded better search directions than did more accurately computed sensitivities for a point. The method selected to avoid overshooting minima on the error surface when updating parameter values with the Gauss-Newton procedure appears for nonlinear models to be more important than the method of sensitivity calculation in controlling regression convergence.

Models, Statistical↗

PubMed1985

Design of anticancer drugs using modeling techniques.

Flexibility of intercalation site geometries within a B-DNA helix was investigated in the twist-shift plane using energy minimization methods. The parameters optimized included sugar conformation, the glycosidic angles and phosphodiester torsion angles. Our calculations show several regions of energetically favorable intercalation geometries in the twist-shift plane. Modeling studies using interactive computer graphics and electrostatic potential surface compatibility provided initial hypotheses for the structures of the drug-DNA complexes. These hypotheses were supported and extended by energy minimizations of these complexes. Binding positions, conformational features and relative minimum binding energies of two anticancer drugs, mitoxantrone and bisantrene, were computed for intercalation complexes with DNA in the theoretically defined intercalation sites. Mitoxantrone intercalates DNA from the minor groove and the side chain OH or NH groups are involved in hydrogen bonds with the main chain phosphate groups of DNA, thereby cross-linking the complementary strands. The hydroxyl groups of mitoxantrone can also participate in hydrogen bonding with phosphate oxygens of another chain, thereby cross-linking DNA helices. Bisantrene intercalates DNA favorably from the major groove and the NH group of the dihydroimidazole ring can participate in hydrogen bonding with the phosphate oxygens of the backbone. These models are consistent with the physicochemical and electron microscopic studies of the interaction of mitoxantrone and bisantrene with DNA. Our results are now being used to guide the design of novel anticancer drugs that should interact with DNA in a manner similar to that proposed for our representative drugs.

Anthracenes↗

PubMed1996

Effect of delivery system on the pharmacokinetics and tissue distribution of bis(di-isobutyl octadecylsiloxy)silicon 2,3-naphthalocyanine (isoBOSINC), a photosensitizer for tumor therapy.

Bis(di-isobutyl octadecylsiloxy)silicon 2,3-naphthalocyanine (isoBOSINC) is a representative of a group of naphthalocyanine derivatives with spectral and photophysical properties that make them attractive candidates for photodynamic therapy (PDT). Tissue distributions were studied in tumor-bearing rats as a function of delivery system and time following administration. The tumor model was an N-(4-[5-nitro-2-furyl]-2-thiazolyl) formamide (FANFT)-induced urothelial cell carcinoma transplanted into one hind leg of male Fischer 344 rats; isoBOSINC was delivered to the rats by intravenous injection of 0.50 mg/kg of body weight as a suspension either in 10% Tween 80 in saline (Tween) or 10% (Cremophor EL + propylene glycol) in saline (Cremophor). The isoBOSINC was isolated from several tissues and organs, as well as tumors and peritumoral muscles and skin. Quantitation was by a high-performance liquid chromatographic technique with detection that utilizes the native fluorescence of the naphthalocyanine derivative. Independent of the delivery system, the dye was retained in tumors at higher concentrations than in normal tissues, except for spleen and liver. The isoBOSINC retention in tumors was high and was vehicle dependent. For Tween, the maximal ratio of dye in tumor versus peritumoral muscle occurred 12 h after injection; for Cremophor, the maximal ratio occurred later, 336 h postinjection. When the drug was delivered in Tween, isoBOSINC in serum showed two compartment kinetics: half-lives of about 2 and 11 h were found for the distribution and the elimination phases, respectively. When Cremophor was the vehicle, the elimination half-life was about 20 h, and one compartment kinetics was observed. The latter findings may explain the generally higher levels of the dye attained by the tissues at later times with Cremophor as the vehicle. An interesting exception was that after 7 and 14 days postinjection in Tween, the levels of dye found in testes were six- to seven-fold higher than those found after Cremophor delivery. Levels of dye were very low or not detectable in the brain. Optimal parameters for PDT of tumors with this novel photosensitizer are clearly time- and vehicle-dependent, and future PDT studies will need to incorporate these modulators.

Animals↗

PubMed2002

Detailed passive cable models of layer 2/3 pyramidal cells in rat visual cortex at different temperatures.

We present detailed passive cable models of layer 2/3 pyramidal cells based on somatic voltage transients in response to brief current pulses at physiological and room temperatures and demonstrate how cooling alters the shape of postsynaptic responses. Whole cell recordings were made from cells in visual cortical slices from 20- to 22-day-old rats. The cells were filled with biocytin and morphologies were reconstructed from three cells which were representative of the full range of physiological responses. These formed the basis for electrotonic models with four electrical variables, namely membrane capacitance (C(m)), membrane resistivity (R(m)), cytoplasmic resistivity (R(i)) and a somatic shunt conductance (G(sh)). Simpler models, with a single value for R(m) and no G(sh), did not fit the data adequately. Optimal parameter values were derived by simulating the responses to somatic current pulses, varying the parameters to give the best match to the experimental recordings. G(sh) and R(m) were badly constrained. In contrast, the total membrane conductance (G(tot)) was well constrained, and its reciprocal correlated closely with the slowest membrane time constant (tau(0)). The models showed close agreement for C(m) and R(i) (ranges at 36 degrees C: 0.78-0.94 microF cm(-2) and 140-170 Omegacm), but a larger range for G(tot) (7.2-18.4 nS). Cooling produced consistent effects in all three model cells; C(m) remained constant (Q(10) = 0.96), R(i) increased (Q(10) = 0.80), whilst G(tot) dropped (Q(10) = 1.98). In terms of whole cell physiology, the predominant effect of cooling is to dramatically lengthen the decay of transient voltage shifts. Simulations suggest that this markedly increases the temporal summation of postsynaptic potentials and we demonstrate this effect in the responses of layer 2/3 cells to tetanic extracellular stimulation in layer 4.

Algorithms↗

PubMedDate not supplied

Electroporation and shock-induced transmembrane potential in a cardiac fiber during defibrillation strength shocks.

Experimental studies have shown that the magnitude of the shock-induced transmembrane potential (Vm) saturates with increasing electric field strength. This study uses a mathematical model to investigate the effects of electroporation and membrane kinetics on Vm in a cardiac fiber. The model consists of the core conductor equation for a one-dimensional fiber, where excitability is represented by the Luo-Rudy dynamic model (1994-1995) and electroporation is described by a membrane conductance that increases exponentially with Vm squared. For shocks delivered during the plateau of an action potential, the model reproduces the experimentally observed saturation of Vm with a root mean square error of 4.27% and a correlation coefficient of 0.9992. For shocks delivered during diastole, the saturation of Vm is qualitatively reproduced even when the sodium and calcium channels are inactivated. Quantitative replication of the response to diastolic shocks is hindered by the choice of electroporation parameters (optimized for shocks delivered during the plateau) and differences in the membrane kinetics between model and experiment. The complex behavior of Vm during large shocks is due to a combination of electroporation, electrotonus, propagation, and active membrane kinetics. The modeling results imply that the experimentally observed saturation of Vm is due to electroporation of the lipid bilayer.

Action Potentials↗

PubMed2002

A new approach to closed-loop linear system identification via a vector autoregressive model.

A new vector autoregressive (VAR) model algorithm is developed for closed-loop identification. The new VAR approach is an extension of a recently developed algorithm, named the optimal parameter search (OPS), thus, we call the new technique VOPS, for vector OPS. Monte Carlo simulations of closed-loop systems were performed to compare the performance of VOPS to the widely utilized vector least squares (VLS) and vector fast orthogonal search (VFOS) approaches. In addition, we examined the effect on parameter estimates obtained via open-loop identification techniques, when using data from closed-loop systems. Comparative results show that both the VOPS and VFOS algorithms produce far more accurate parameter estimates than does the VLS. Furthermore, open-loop identification via univariate OPS and to a lesser extent univariate FOS for closed-loop systems, does not adversely affect the accuracy of the parameter estimates. An open-loop identification via the univariate least-squares method for closed-loop systems showed the most deleterious effect on the accuracy of the parameter estimates. In addition to developing the VOPS algorithm, we also developed approaches termed constrained OPS (COPS) and constrained FOS (CFOS). For closed-loop systems considered in this paper, both COPS and CFOS resulted in more accurate parameter estimates (less biased and more efficient) than did VLS, VFOS, and VOPS.

Algorithms↗