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PubMed2001

Optimization of the immobilization parameters and operational stability of immobilized hydantoinase and l-N-carbamoylase from Arthrobacter aurescens for the production of optically pure l-amino acids.

2The immobilization parameters were optimized for the hydantoinase and the L-N-carbamoylase from Arthrobacter aurescens DSM 3747 or 3745, respectively. To optimize activity yields and specific activities for the immobilization to Eupergit C, Eupergit C 250 L, and EAH-Sepharose wild-type, recombinant and genetically modified ('tagged') enzymes were investigated concerning the influence of the protein concentration, the kind of support and the immobilization method. For both enzymes, the use of the recombinant proteins resulted in enhanced specific activities especially when using a hydrophilic support for immobilization such as Sepharose. In the case of a genetically modified hydantoinase carrying a His(6)-tag, affinity coupling led to a loss of activity of higher than 80%. Both enzymes were significantly stabilized by immobilization: In packed bed reactors, Eupergit C 250 L (NH(2))-immobilized hydantoinase and EAH-Sepharose-immobilized L-N-carbamoylase showed half-life times of approx. 14000 and 900 hours, respectively. Together with specific activities of the immobilized enzymes of 2.5 U/g wet carrier (hydantoinase) and 10 U/g wet carrier (L-N-carbamoylase) the newly developed biocatalysts are sufficient to fulfill industrial requirements.In comparison to the free enzymes, temperature and pH-optima were increased by 10 degrees C and one pH unit, respectively, after immobilization. The pH and temperature optima of the hydantoinase (L-N-carbamoylase) were determined to be pH 8.5-10 (pH 9.5) and 45-60 degrees C (60 degrees C).In order to provide sufficient amounts of biocatalyst for the process development in mini plant scale, a 50 fold scale-up of the optimized immobilization procedure was carried out for both enzymes. Because of the overlapping optima, both immobilized enzymes can be operated together in one reactor.

Journal Article↗

PubMed1999

Determination of the ratio of D- and L-amino acids in brewing by an immobilised amino acid oxidase enzyme reactor coupled to amperometric detection.

To determine the quantity of free amino acids, the D- and L-forms separately, is an important task in modern nutritional studies. The aim of our present work was to develop rapid, routine methods for fast determination of the different forms of free amino acids. We utilized two enzymes (L-amino acid oxidase, D-amino acid oxidase) with broad specificity. In our home-made reactors, the enzymes were immobilized in a thin-layer Plexi-cell on natural protein membrane. The enzyme-cell was built into a FIA system and the hydrogen peroxide generated during the enzymatic reaction was determined by an amperometric detector. The electrode potential was fixed at +100 mV. The parameters for the biochemical and electrochemical reactions were optimized in each case. The optimal pH value for measuring L- and D-amino acids was found ca. 8.8 and 9.5, respectively. The LAO reactor could be used for more than 900 measurements, while the DAO reactor for about 1000 measurements. The working concentration range was between 0.1-3 and 0.2-3 mM, respectively. The same standard solution (L- and D-Methionine, 1 mM) was injected 25 times sequentially and the standard deviations were 2 and 2.7%, respectively. After determining the optimal parameters, the specificity of the immobilized enzyme preparations towards different amino acids and in samples from different stages of brewing was investigated.

Amino Acid Oxidoreductases↗

PubMed1989

The evolution strategy--a search strategy used in individual optimization of electrical parameters for therapeutic carotid sinus nerve stimulation.

Optimization problems, arising in the search for parameters and/or techniques of functional electrostimulation (FES), disproportionally increase when multiple electrodes, electrode configurations, electrical parameters, and stimulation modes may be applied. When computational or investigational effort precludes systematic studies in FES, we propose to apply and evaluate Rechenberg's evolution strategy, which in technical use and numerical optimization has been valid in comparison to more traditional methods. This strategy implements mutation and selection processes in analogy to biological evolution. The effect of combined multiple input variables on a quality function (Q) is experimentally evaluated. The actual computed value of Q serves as a selection criterion for those input variable combinations which lead Q to approach a target value (maximization), similar to a hill-climbing procedure. In radiofrequency controlled, therapeutic electrical carotid sinus nerve stimulation (CSNS), we varied (mutated) combinations of pulse frequency and pulse amplitude parameters, according to the evolution strategy, in individual patients. CSNS lowers blood pressure and decreases heart rate. Q was computed from blood pressure and heart rate responses to CSNS. The strategy individually optimized electrical parameters to achieve large depressor responses upon CSNS. Although, in contrast to technical usage, only two input variables were investigated, and biomedical experience with the evolution strategy is limited so far, its potential use in other fields of FES, especially when more input variables are to be optimized, is discussed and encouraged.

Algorithms↗

PubMed1999

High-Resolution Infrared Spectrum of nu5, nu4, nu3, nu4 + nu5, and 2nu4 Band Systems of Deuterobromoacetylene.

High-resolution vibration-rotation spectra of gas-phase deuterobromoacetylene have been recorded in the 240-990 cm-1 infrared region. The analyzed band systems are rich in hot bands and have a high density of lines. Five band systems and a total of 124 vibration-rotation bands of the isotopic species DCC79Br and DCC81Br have been rotationally analyzed. Accurate rotational parameters and vibrational wavenumbers for 33 vibrational states of each species have been obtained from the rotational analysis. l doubling and rotational l resonance have been observed on some states and the respective resonance parameters have been obtained through nonlinear least-squares optimization. A Fermi resonance block model with perturbation terms has been used for the analysis of the vibrational states. With optimized parameters, the model produces root-mean-square deviations of observed - calculated wavenumbers of about 0.3 cm-1 for both isotopic species. Copyright 1999 Academic Press.

Journal Article↗

PubMed1997

[Radiofrequency ablation of the liver in vitro: increasing the efficacy by perfusion probes].

PURPOSE: Percutaneous radiofrequency interstitial thermal ablation is a new method in local tumour therapy. The aim of this study was to define the relations between the variable parameters and local efficacy in vitro and to evaluate optimal parameter combinations for this system. Furthermore, we studied the feasibility of increasing the volume of destroyed tissue using perfusion electrodes. MATERIAL AND METHODS: Thermal lesions were produced with radiofrequency in ex vivo pig livers. In separate experiments the parameters wattage (5-30 watts), exposure time (1-15 minutes) and tip exposure were varied. The resulting areas of tissue necrosis coagulation were measured; in 30 of 90 cases the macroscopic findings were compared to the histological findings. RESULTS: Lesion size correlated with tip exposure, wattage and procedure duration up to 10 minutes. For a tip exposure of 2 cm (3 cm) the maximal lesion volume was 18.8 cm3 (33.2 cm3) at a wattage of 20 watts (30 watts) and a procedure duration of 10 minutes. The maximal cross- (length-) diameter of these lesions was 3 cm (4 cm) for 2 cm tip exposure and 3.6 cm (4.9 cm) for 3 cm tip exposure. CONCLUSION: The parameters wattage, procedure duration and tip exposure affect the size of lesions created with radiofrequency under ex vivo conditions. Perfusion electrodes make it possible to produce larger lesions than described for non-perfused electrodes. The ablation of hepatic neoplasms up to a size of 3 cm seems to be possible with a single electrode.

Animals↗

PubMed2001

A refined prediction method for gel retardation of DNA oligonucleotides from dinucleotide step parameters: reconciliation of DNA bending models with crystal structure data.

The development and assessment of a prediction method for gel retardation and sequence dependent curvature of DNA based on dinulcleotide step parameters are described. The method is formulated using the Babcock-Olson equations for base pair step geometry (1) and employs Monte Carlo simulated annealing for parameter optimization against experimental data. The refined base pair step parameters define a stuctural construct which, when the width of observed parameter distributions is taken into account, is consistent with the results of DNA oligonucleotide crystal structures. The predictive power of the method is demonstrated and tested via comparisons with DNA bending data on sets of sequences not included in the training set, including A-tracts with and without periodic helix phasing, phased A4T4 and T4A4 motifs, a sequence with a phased GGGCCC motif, some "unconventional" helix phasing sequences, and three short fragments of kinetoplast DNA from Crithidia fasiculata that exhibit significantly different behavior on non-denaturing polyacrylamide gels. The nature of the structural construct produced by the methodology is discussed with respect to static and dynamic models of structure and representations of bending and bendability. An independent theoretical account of sequence dependent chemical footprinting results is provided. Detailed analysis of sequences with A-tract induced axis bending forms the basis for a critical discussion of the applicability of wedge models,junction models and non A-tract, general sequence models for understanding the origin of DNA curvature at the molecular level.

Base Sequence↗

PubMed1985

[The dynamic property of glucagon secretion in response to blood glucose--analysis with the aid of the control theory using an artificial endocrine pancreas].

The precise dynamic property of glucagon secretion in response to glucose concentration has not yet been elucidated, since in in vitro studies using pancreatic islets, the co-existence of pancreatic B cells modifies the mode of glucagon secretion, and in vivo studies, the exogenous insulin administration greatly affects pancreatic A cell function. In this study, to clarify the dynamic property of glucagon secretion in response to glucose concentration and the ability of glucagon to raise hepatic glucose production, an artificial endocrine pancreas was employed as a research tool. Our originally developed artificial endocrine pancreas prepared the glucagon and/or glucose infusion algorithm as the counterregulatory system. The principle of glucagon and glucose infusion algorithm is set as the proportional plus derivative modes of action to blood glucose concentration with time delay constant, as follows; Gn.I.R.(t) = Gp[BGp - BG(t - tau)] + Gd[-delta BG(t - tau)] + Gc G.I.R.(t) = Cp[BGp - BG(t - tau)] + Cd[-delta BG(t - tau)] where Gn.I.R.(t) and G.I.R.(t) are glucagon infusion rate and glucose infusion rate, respectively. BGp is the projected value of blood glucose concentration, and BG(t) and delta BG are blood glucose concentrations and the rate of change in blood glucose concentration at time t respectively. Gp and Gd are coefficients for glucagon infusion, and Cp and Cd are those for glucose infusion. Gc is the constant for basal glucagon supplementation. tau(min) is the time delay constant for glucagon and glucose infusion. In a depancreatized dog, the blood glucose concentration was maintained at the normoglycemic level with intraportal insulin infusion using this artificial endocrine pancreas for at least one hour, then hypoglycemia was induced by iv bolus insulin injection (0.1 U/kg). Glucagon was infused intraportally or glucose was infused into the peripheral vein when the counterregulatory system was operated according to each of these algorithm by variously changing the parameters. In the intraportal glucagon infusion algorithm, with the optimal parameters based on proportional plus derivative modes of action with a 10-min time delay (Gp/Gd/Gc/tau = 0.2/0.4/0.6/10), both the blood glucose response curves and plasma glucagon profiles simulated those seen in normal dogs. On the other hand, glucagon infusion based only on the proportional action failed to simulate the blood glucose response and plasma glucagon profile of normal dogs. When glucose was infused on the basis of the proportional action with a 20-min time delay (Cp/Cd/tau - 0.2/0/20), the insulin-induced hypoglycemia in depancreatized dogs could be restored to normoglycemia in the sa

Animals↗

PubMed2002

Modeling, optimization and experimental assessment of continuous L-(-)-carnitine production by Escherichia coli cultures.

In a previous paper Cánovas et al. (Biotechnol Bioeng 2002;77:764-775) presented a model for L-(-)-carnitine production using Escherichia coli O44 K74, in a cell-recycle bioreactor for the biotransformation of crotonobetaine into L-carnitine. In this work we optimize this biotechnological setup and experimentally verify the predicted optimal parameter profiles. Provided with a reliable and robust S-system description of the cell-bioreactor combined system, we applied the Indirect Optimization Method described by Torres et al. (Biotechnol Bioeng 1997;55(5):758-772; Food Technol Biotechnol 1998;36(3):177-184). This optimization approach provides different parameter value profiles, all of which are compatible with the cell physiology and the bioreactor operating conditions, that yield increased rates of L-(-)-carnitine production. Three parameters were seen to be of critical importance for maximizing L-(-)-carnitine production: the dilution rate, the initial crotonobetaine concentration, and the carnitine dehydratase activity. When the first two were changed in the experimental setup, there was a 74% increase in the L-(-)-carnitine production rate, performance that was in close agreement with the predictions of the model. In accordance with the optimized solution, a further improvement (90% increase in the L-(-)-carnitine production rate) could be attained by over-expressing up to 5 times the carnitine dehydratase basal activity. Thus the optimization approach shown herein provides experimental evidence of a new strategy which demonstrates the possible variables that can be subjected to modifications compatible with the cell physiology and bioreactor operating conditions, and which are able to yield increased rates of L-(-)-carnitine production.

Alcohol Oxidoreductases↗

PubMed1979

Single-tissue modeling of decompression schedules.

This paper deals with the development of a single-tissue model that simulates the uptake and elimination of inert gases by the body of a diver. The model utilizes an effective single tissue with different uptake and elimination time constants to account for the asymmetrical behavior of multiple-tissue human body models. The parameters of this effective tissue are selected according to an optimal strategy that minimizes safe deviation from the decompression requirements recommended by safe practice. The developed strategy is general in nature and can be readily applied to select the optimal parameters for a single-tissue model suitable for any dive regimen on air or mixed gas. As an illustration, the procedure is used to select the optimal tissue that best fits the Standard Air Decompression Tables recommended by the U.S. Navy. The results obtained are in close and safe agreement with the requirements of the U.S. Navy, and consistently fall in the range between the U.S. Navy and the Royal Navy tables.

Decompression↗

PubMed1999

Comparison of five methods for finding conserved sequences in multiple alignments of gene regulatory regions.

Conserved segments in DNA or protein sequences are strong candidates for functional elements and thus appropriate methods for computing them need to be developed and compared. We describe five methods and computer programs for finding highly conserved blocks within previously computed multiple alignments, primarily for DNA sequences. Two of the methods are already in common use; these are based on good column agreement and high information content. Three additional methods find blocks with minimal evolutionary change, blocks that differ in at most k positions per row from a known center sequence and blocks that differ in at most k positions per row from a center sequence that is unknown a priori. The center sequence in the latter two methods is a way to model potential binding sites for known or unknown proteins in DNA sequences. The efficacy of each method was evaluated by analysis of three extensively analyzed regulatory regions in mammalian beta-globin gene clusters and the control region of bacterial arabinose operons. Although all five methods have quite different theoretical underpinnings, they produce rather similar results on these data sets when their parameters are adjusted to best approximate the experimental data. The optimal parameters for the method based on information content varied little for different regulatory regions of the beta-globin gene cluster and hence may be extrapolated to many other regulatory regions. The programs based on maximum allowed mismatches per row have simple parameters whose values can be chosen a priori and thus they may be more useful than the other methods when calibration against known functional sites is not available.

Animals↗

PubMed1980

Split-filter computed tomography: a simple technique for dual energy scanning.

A simple technique for obtaining dual energy information from a single computed tomography (CT) scan is described. The method involves filtering the two halves of the X-ray fan beam differently. It is shown that during a 360 degree scan, this split-filtration geometry results in dual energy noise analysis, valid for polychromatic X-ray beams, is presented and used to determine the optimal parameters for the split-filter scans. Using the theoretically optimal split-filter design installed on a commercial CT scanner, photoelectric and Compton images of a head phantom were obtained. Recombination of the photoelectric and Compton components of attenuation at arbitrary energies demonstrates the ability to remove spectral artifacts from conventional CT images.

Calibration↗

PubMed2001

Routine cervical spine radiography for trauma victims: Does everybody need it?

OBJECTIVE: The purpose of this study was to evaluate the indication for routine cervical spine radiography in trauma patients. METHODS: Prospective analysis of radiologic and clinical findings was performed during a 5-year period. Patients suitable for a clinical decision rule were reviewed separately. RESULTS: Of the 1,757 consecutive patients included in the study, 38 were diagnosed with a cervical spine injury. Of the 599 patients suitable for the clinical decision rule, 62 had midline cervical tenderness, including 2 with cervical spine injury. No additional cervical spine injuries were found during follow-up. CONCLUSION: It is within good practice, and it is also cost-effective, to obtain a cervical spine radiograph only on clinical parameters in trauma patients with no apparent bodily trauma and optimal parameters. With this clinical decision rule, 30.6% of all cervical spine series were redundant, and no (occult) spinal fractures would have been undetected.

Adolescent↗

PubMed2000

The boundary element method in the forward and inverse problem of electrical impedance tomography.

In this paper, a new formulation of the reconstruction problem of electrical impedance tomography (EIT) is proposed. Instead of reconstructing a complete two-dimensional picture, a parameter representation of the gross anatomy is formulated, of which the optimal parameters are determined by minimizing a cost function. The two great advantages of this method are that the number of unknown parameters of the inverse problem is drastically reduced and that quantitative information of interest (e.g., lung volume) is estimated directly from the data, without image segmentation steps. The forward problem of EIT is to compute the potentials at the voltage measuring electrodes, for a given set of current injection electrodes and a given conductivity geometry. In this paper, it is proposed to use an improved boundary element method (BEM) technique to solve the forward problem, in which flat boundary elements are replaced by polygonal ones. From a comparison with the analytical solution of the concentric circle model, it appears that the use of polygonal elements greatly improves the accuracy of the BEM, without increasing the computation time. In this formulation, the inverse problem is a nonlinear parameter estimation problem with a limited number of parameters. Variants of Powell's and the simplex method are used to minimize the cost function. The applicability of this solution of the EIT problem was tested in a series of simulation studies. In these studies, EIT data were simulated using a standard conductor geometry and it was attempted to find back this geometry from random starting values. In the inverse algorithm, different current injection and voltage measurement schemes and different cost functions were compared. In a simulation study, it was demonstrated that a systematic error in the assumed lung conductivity results in a proportional error in the lung cross sectional area. It appears that our parametric formulation of the inverse problem leads to a stable minimization problem, with a high reliability, provided that the signal-to-noise ratio is about ten or higher.

Algorithms↗

PubMed2002

Piezoelectrically actuated flextensional micromachined ultrasound transducers--I: theory.

This series of two papers considers piezoelectrically actuated flextensional micromachined ultrasound transducers (PAFMUTs) and consists of theory, fabrication, and experimental parts. The theory presented in this paper is developed for an ultrasound transducer application presented in the second part. In the absence of analytical expressions for the equivalent circuit parameters of a flextensional transducer, it is difficult to calculate its optimal parameters and dimensions and difficult to choose suitable materials. The influence of coupling between flexural and extensional deformation and that of coupling between the structure and the acoustic volume on the dynamic response of piezoelectrically actuated flextensional transducer are analyzed using two analytical methods: classical thin (Kirchhoff) plate theory and Mindlin plate theory. Classical thin plate theory and Mindlin plate theory are applied to derive two-dimensional plate equations for the transducer and to calculate the coupled electromechanical field variables such as mechanical displacement and electrical input impedance. In these methods, the variations across the thickness direction vanish by using the bending moments per unit length or stress resultants. Thus, two-dimensional plate equations for a step-wise laminated circular plate are obtained as well as two different solutions to the corresponding systems. An equivalent circuit of the transducer is also obtained from these solutions.

Computer Simulation↗

PubMed1984

Optimization of an hydroxyapatite adhesion assay for Streptococcus sanguis.

Previous studies have compared the adhesion of [3H]thymidine-labeled Streptococcus sanguis to saliva-coated hydroxyapatite (SHA) and buffer-coated hydroxyapatite (HA) beads. Although the hypotonic buffer used in these assays was adjusted to simulate saliva, it does not necessarily provide the optimal parameters for the quantitative estimate of adhesion under in vitro conditions. Optimization is necessary to provide the maximum sensitivity of the assay for detecting the effects of various salivas as well as for quantitating the effect of environmental growth conditions on the adhesion of S. sanguis to SHA and HA. A major distinction between the adhesion of S. sanguis to SHA and HA was observed when the bacterial concentration was varied. At high cell concentrations, the number of cells adhering to SHA was twice the number adhering to HA. Such differences were not detected at low cell concentrations. The optimal pH for the adsorption to both SHA and HA was 6. Changes in the ionic strength or addition of mono- or divalent cations found in saliva had little effect on adhesion to HA. In contrast, high concentrations of monovalent cations inhibited adhesion to SHA. Anions such as carbonate, chloride, and sulfate did not have specific effects on adhesion, whereas acetate inhibited adhesion to both SHA and HA. Fluoride inhibited adhesion to both SHA and HA, suggesting an interaction between fluoride and hydroxyapatite. These results indicated that 2 mM phosphate buffer at a pH of 6 containing 5 mM KCl and 1 mM CaCl2 was the optimal buffer for studying the in vitro adhesion of S. sanguis to SHA.

Durapatite↗

PubMed1998

Spatial and temporal matching of receptive field properties of binocular cells in area 19 of the cat.

The spatial and temporal properties of single neurons were investigated in area 19 of the cat. We evaluated the matching of binocular receptive field properties with regard to the respective strength of the ipsilateral and contralateral inputs. Results indicate that most cells in area 19 are well tuned to spatial and temporal frequencies and exhibit relatively low contrast threshold (mean=6.8%) when assessed using optimal parameters and tested through the dominant eye. Spatial resolution (mean=0.75 c/degree), optimal spatial frequencies (mean=0.16 c/degree) were relatively low and spatial bandwidths (mean=2.1 octaves) were broader as compared to those of cells in area 17 but comparable to those of cells in other extrastriate areas. On the other hand temporal resolution (mean=10.7 Hz), optimal temporal frequency (mean=4.5 Hz) and temporal bandwidths (mean=2.9 octaves) were higher and broader than in primary visual cortex. A significant relationship exists between most of the cell's properties assessed through either eye. For some parameters, such as spatial and temporal resolution, ocular dominance was shown to be significantly related to the extent of matching between the two eyes. For these parameters, binocular cells that exhibited a balanced ocular dominance were generally well matched with regard to the receptive field properties of each eye whereas the largest mismatches were found in cells that were more strongly dominated by one eye. These results suggest that visual input contributes to the activation of cells in area 19 in a redundant manner, possibly attesting to the multiplicity of parallel pathways to this area in the cat.

Animals↗

PubMedDate not supplied

[Effect of the conditions of isolation of free and bound ligands on the accuracy of the radioimmunoassay of steroids].

Testosterone radioimmunoassay (RIA) was used to analyse the factors which influenced the precision and accuracy of steroid RIA. Selection of optimal parameters (concentrations of activated charcoal and of the radioactive steroid) was necessary in order to reduce the influence of non-specific binding and error of fractionation on the RIA results. The scheme for estimation of the optimal values of these parameters is described.

Charcoal↗

PubMed1975

Studies on the feasibility of urinary bladder evacuation by direct spinal cord stimulation. I. Parameters of most effective stimulation.

In 30 female dogs, spinal cord stimulation was tested to find the optimal parameters producing good voiding and, hopefully, total bladder emptying. Twelve different electrodes or application modifications were examined. Variable frequencies, stimulus duration, and voltages were applied. Search of the site of highest detrusor response (micturition center) was easily identifiable. It became obvious that electrode design was not of critical importance: the responses were basically the same whether we used the wrap-around electrode, which stimulates an entire level, or the coaxial electrode which theoretically limits the current spread around its minute exposed tip. The stimulation parameters that gave the best results were: 2 to 5 volts; frequency: 10 to 15 cps; duration 1 msec. The site of highest detrusor response was round vertebral level L5 1/2, corresponding to cord level S2-4. Generally, no voiding was obtained because of too high sphincteric contraction during stimulation; it appeared that a sphincteric center overlaps the micturition center in either direction. To achieve successful voiding during stimulation, a separation of these two centers with selective stimulation of the detrusor center is necessary, eleiminating sphincteric contraction.

Animals↗