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Noninvasive metabolic analysis of eye bank corneas: a magnetic resonance spectroscopic study.

Nondestructive, noninvasive metabolic analysis of corneal donor tissue preserved in modified McCarey-Kaufman medium was achieved using phosphorus-31 magnetic resonance spectroscopy (31 P MRS). Clear corneas (n = 10) with minimal Descemet's folds and an endothelial cell density of 2990 +/- 166 cells/mm2 from donors aged 26 to 89 years were used. Relative amounts of low- and high-energy phosphatic metabolites were quantitated during a 1-h period using the integral of the 31P spectrum. The following indicators of the tissue's energy status were determined: (1) 31P spectral energy modulus (ratio of high- to low-energy metabolites, average 0.95 +/- 0.14); (2) adenosine triphosphate/inorganic orthophosphate (ATP/Pi) ratio, 1.97 +/- 0.55; (3) sugar phosphate/inorganic orthophosphate (SP/Pi) ratio, average 1.07 +/- 0.12; and (4) intracorneal pH, calculated from the resonance shift position of inorganic orthophosphate, average 7.28 +/- 0.05. The 31P MRS technique measures key corneal metabolic processes (maintenance of the high-energy phosphate complement, interrelationship of inorganic orthophosphate and its bioesters, and pH). This study provides baseline data for eventually determining optimal parameters of eye bank corneal tissues and for evaluating the metabolic status, or health, of the cornea.

Adenosine Triphosphate

DNA-mediated gene transfection into primary cultures of adult mouse keratinocytes.

An efficient and reproducible technique for the transfection of primary cultures of adult mouse keratinocytes has been developed. The procedure involves culturing the primary adult mouse epidermal cells at 32 degrees C in an enriched media until they reach 70 to 95% confluency, followed by transfection with exogenous DNA in a low potassium environment. Using chloramphenicol acetyl transferase (CAT) transient gene expression assays and various strong viral promoter/CAT constructs, the transfection procedure was optimized for media formulation, plasmid DNA concentration, carrier DNA concentration, incubation temperature, incubation period, and cell density. Optimized parameters include the use of 6 micrograms plasmid DNA and 10 micrograms pUC19 carrier DNA per 60-mm tissue culture dish. Since primary keratinocytes undergo a well-characterized pattern of differentiation in vitro in response to extracellular calcium concentrations, this transfection procedure should provide a useful model in which to study both tissue- and differentiation-specific gene expression.

Animals

Use of S1 nuclease and formamide in combination for the reassociation studies on GC-rich DNA.

The optimal parameters in the use of nuclease S1 in DNA reassociation kinetics in the presence of formamide have been determined. The conditions are especially suitable for the study of DNA rich in mole percent GC. A 10-fold dilution of the reassociation samples leading to a decrease in both NaCl and formamide concentrations, consequently resulting in a lowering of Tm by only 1.5 degree C, and the S1 digestion at temperatures identical to the reassociation assay in order to retain the stability of the duplex, are two important aspects of this system. Under these conditions, the kinetics of reassociation followed the theoretically predicted pattern, while the earlier reported methods have shown lower values.

Chemical Phenomena

CO binding to mitochondrial mixed valence state cytochrome oxidase at low temperatures.

The kinetics and thermodynamics of the reaction of mixed valence state membrane-bound cytochrome oxidase with CO over the 178-203 K range has been studied by multichannel optical spectroscopy at three wavelength pairs (444-463 nm in the Soret region, and 590-630 and 608-630 nm in the alpha region) and analysed by non-linear optimization techniques. As in the case of the fully reduced membrane-bound cytochrome oxidase-CO reaction (Clore, G.M. and Chance, E.M. (1978) Biochem J. 175, 709-725), the normalized progress curves at the three wavelength pairs are significantly different indicating, on the basis of Beer's law, the presence of a minimum of three optically distinct species. The only model that satisfies the triple statistical requirement of a standard deviation within the standard error of the data, a random distribution of residuals and good determination of the optimized parameters, is a two species sequential mechanism: flash photolysis of the mixed valence state cytochrome oxidase-CO complex (species IIMC) yields unliganded mixed valence state cytochrome oxidase (species EM) and free CO which then recombine to form species IMC; species IMC is then converted into species IIMC. All the thermodynamic parameters describing the model are calculated and compared to those obtained for the fully reduced membrane-bound cytochrome oxidase-CO reaction (Clore and Chance (1978) Biochem. J. 175, 709-725). Although there are some qualitative similarities in the kinetics and thermodynamics of the reactions of mixed valence state (alpha 23+Cu+B.ALPHA 3+Cu2+A) and fully reduced (a3 2+Cu B + . a2+Cu A+) cytochrome oxidase with CO, there are large and significant quantitative differences in zero-point activation energies and frequency factors; over the temperature range studied, the mixed valence state cytochrome oxidase-CO reaction is found to proceed at a significantly slower rate than the fully reduced cytochrome oxidase-CO reaction. These differences indicate that changing the valence states of cytochrome a and CuA has a significant effect on the CO binding properties of cytochrome a 3 and possibly CuB.

Carbon Monoxide

A re-evaluation of the low-temperature kinetics of the reaction of fully reduced mitochondrial cytochrome oxidase with carbon monoxide and the spectral characterization of species Ic in the Soret and visible regions.

The kinetics of the reaction of fully reduced membrane bound cytochrome oxidase with CO following photolysis of the fully reduced cytochrome oxidase-CO complex habe been re-examined by re-analysing the data of Clore and Chance (1978) Biochem. J. 175, 709-725) at six temperatures in the 178-203 K range simultaneously at only a single wavelength pair, 444-463 nm. The choice of the 444-463 nm wavelength pair was based on the fact that the absorbance change produced at 444-463 nm on photolysis of the CO complex is sufficiently large and the separation between monitoring and reference wavelengths sufficiently small to render the effects of any possible time dependent scattering changes insignificant. On the basis of our analysis only a two step mechanism (Model 1 of Clore and Chance (1978) Biochem. J. 175, 709-725) satisfies the triple requirement of a S.D. within the standard error of the data, a random distribution of residuals and good determination of the optimized parameters. The single step mechanism of De Fonseka and Chance (1978) Biochem. J. 175, 1137-1138) fails to satisfy all three requirements. The pure difference spectra of species Ic minus E, E minus IIc and Ic minus IIc are calculated from the computed kinetics of the individual species and repetitive slow wavelength scanning difference spectra (reaction sample minus the CO complex) taken during the course of the reaction of fully reduced cytochrome oxidase with CO at 176 K.

Animals

NL-FIT: a microcomputer program for non-linear fitting.

A program for a microcomputer (HP 85) has been written using BASIC language. Given the analytical form of a model y: R leads to R the program computes the optimal parameters for non-linear least-squares fitting and gives information for its statistical evaluation. The program uses the Gauss-Newton algorithm with Marquardt's modification and other tricks. During the run a message is displayed if there is a high correlation between one or more couples of parameters to establish an interactive dialogue with the user on critical problems. The authors' aim was to make the program sufficiently quick and easy to use, efficient for a wide class of models and robust enough to deal with bad starting parameters.

Computers

On the estimation of pulmonary blood flow from CO2 production time series.

It may be possible to estimate a nominal pulmonary blood flow (Q) during an exercise stress test via the algorithm used to estimate breath-by-breath alveolar CO2 production. Recently it has been demonstrated that by relating breath-to-breath fluctuations in alveolar CO2 production to breath-to-breath fluctuations in end-tidal CO2, an optimizing parameter related to Q can be determined that can be used to process the CO2 production fluctuations and minimize their variation. However, the reported values of Q using this procedure appear to be biased low. Using a computer simulation of gas exchange, we demonstrate that the estimate of Q is biased low when the nominal lung volume used in the alveolar gas exchange algorithm is too large. Furthermore, alveolar CO2 transport is determined by an integral of alveolar CO2 over the breath time and, thus, is a path-dependent quantity. The use of end-tidal CO2 fluctuations to approximate fluctuations in this integral contributes to an error in the estimation of Q which yields estimates that are biased low. Alternatively, the use of mean alveolar CO2 fluctuations yield more appropriate Q estimates. These results suggest practical implications for estimating effective pulmonary blood flow during an exercise stress test by using breath-to-breath estimates of mean alveolar CO2.

Blood Flow Velocity

Computer-assisted dynamic integration of multiple medical thesauruses.

We have previously described a user-interactive rule-based computer program (Dyna-SaurI) designed for dynamic thesaurus integration, and demonstrated its efficacy on integrating dermatological subsets of the MeSH and SNOMED thesauruses. In the present study, we have refined our rules for merging and mapping multiple thesauruses and tested these rules. We then applied them with a set of optimized parameters to the integration of a third thesaurus, a subset of the International Coding Index for Dermatology, with the Integrated MeSH-SNOMED thesaurus. The parameter changes resulted in improved ranking of more specific and conceptually closer terms.

Abstracting and Indexing

Surface coil phosphorus-31 nuclear magnetic resonance studies of the intact eye.

The feasibility of employing the surface coil probe technique for the non-invasive study of ocular tissue metabolism by phosphorus-31 nuclear magnetic resonance spectroscopy (31P NMR) in enucleated bovine, rabbit, human and rat globes is demonstrated. An assessment of individual phosphorus-metabolite contributions from ocular tissues, including the cornea, lens and iris, to the overall 31P NMR spectrum (NMR spectral acquisition parameters optimized for the lens region of the globe) was accomplished through the combination of surgical ablation and difference spectroscopy. The NMR measurements also provided tissue pH values for the lens and cornea. The strengths and limitations of the surface coil NMR method, which is particularly appropriate for in vivo metabolic studies of ocular tissues such as the lens, are discussed.

Adenosine Triphosphate

Modification, optimization and simplification of the spot ELISA technique for the enumeration of cells secreting anti-hapten antibodies.

We describe here a modification of the spot ELISA assay for the detection and enumeration of antibody-secreting cells. The modification increases sensitivity, convenience and simplicity while optimizing parameters of time, temperature and reagents. Laboratory reagents and 96 well microtiter plates commonly used for the standard ELISA assay are adapted for use in the spot ELISA assay. Special emphasis is placed on the detection of anti-hapten IgM or IgG production by splenocytes in response to stimulation by a range of doses of antigen. In addition, two procedures are described whereby the spot ELISA assay is altered to yield a measurable optical density: (1) the blue precipitate obtained in the spot ELISA assay is dissolved and the optical density of the resultant blue color is measured, and (2) a standard ELISA substrate is substituted for the spot ELISA substrate to yield a colored solution rather than a precipitate. Both of these are shown to correlate with spot numbers and can therefore supplement or substitute for the enumeration of spots (produced by antibody-secreting cells), thus speeding and mechanizing the assay.

Animals

Fetal whole-body permeability--surface area product and reflection coefficient for plasma proteins.

The transcapillary distribution of protein between the vascular and interstitial spaces largely depends on the protein permeability characteristics of the capillary membranes, i.e., the reflection coefficient and permeability - surface area product for plasma proteins. Our purpose was to estimate whole-body values for these parameters in chronically catheterized, near-term fetal sheep. Average experimental changes in plasma protein concentration, blood volume and vascular pressures following fetal hemorrhage or intravascular saline infusion were analyzed using mathematical modeling and parameter optimization techniques. The mathematical model consisted of three interacting compartments: (1) fetal blood, (2) fetal interstitium, and (3) maternal blood. We found that the mean fetal whole-body protein reflection coefficient averaged 0.61 following hemorrhage and 0.80 after intravascular saline infusions. The protein permeability - surface area product averaged 0.36 ml/min/kg for the hemorrhages and 0.70 ml/min/kg following saline infusions. The major findings are that the fetus has (1) a permeability - surface area product averaging 15 times that of the adult, (2) a protein reflection coefficient approximately 70 to 90% of adult values, and (3) the ability to decrease its capillary surface area following hemorrhage.

Animals

Impedance of the lower respiratory system in ducks measured by forced oscillations during normal breathing.

The lower respiratory system of 10 conscious Pekin ducks breathing normally was subjected to superimposed oscillations of 1.3 to 16 Hz by a small volume piston pump. Induced sinusoidal flow (VO) and pressure (PO) signals were measured in late expiration, when the gas flow ventilated by the animal was minimum. The modulus of the respiratory impedance was calculated as the ratio PO/VO. The values obtained at the various oscillatory frequencies were compared to those predicted on the basis of a series mechanical network model consisting of resistive, inertial and compliant elements (RIC) using a least squares non linear regression method. The experimental data fitted well the frequency response of a simple RIC mathematical model. After correcting for the effects of the endotracheal tube, the mean values +/- SE of the optimized parameters were: resistance 4.8 +/- 0.4 cm H2O . L-1 . sec; inertance: 0.05 +/- 0.01 cm H2O . L-1 . sec2; compliance: 7.7 +/- 0.5 ml . cm H2O-1; natural frequency: 8.0 +/- 0.4 Hz. It is concluded that: (1) the lower respiratory system in ducks can be closely modeled by a RIC mechanical series network model; (2) the forced oscillation method can be used to investigate avian mechanics of breathing, with the birds awake and breathing spontaneously.

Airway Resistance

Psychosocial adjustment after renal retransplants.

Twenty-four patients who underwent more than one kidney transplant were interviewed to assess their psychosocial adaptation. Of these 24, we found 15 patients with a functioning kidney and 9 patients with a nonfunctioning transplant who were back on hemodialysis. In both groups, approximately 50% of the patients had life satisfaction ratings of "good" and "very good." The groups differed in two respects. Those with a functioning kidney had a higher employment rate and a better sexual functioning than the group on dialysis. We found many similarities in all other scored parameters (optimism, object relationships, coping devices, psychologic functioning). Denial was commonly used by most of the patients, but the group of patients who scored "good" and "very good" on life satisfaction more frequently utilized other coping devices, such as altruism, "feeling chosen," inner control, and activity than patients with "fair" or "poor" life satisfaction.

Adaptation, Psychological

A simple algorithm for the solution of the multiple dosing problem.

A system theoretical approach to the general multiple dosing problem is discussed [1], which is in turn reduced to a classical parameter optimization problem. An efficient numerical algorithm based on the cyclic coordinate search method to solve the latter is explained. A flexible and user-friendly program written in standard FORTRAN which is suitable for this problem is introduced. Finally, the problem is applied to the dopaminergic substitution problem in Parkinson's disease, and the necessary input data as well as the resulting output is displayed in a sample run.

Computers

GGOPT: an unconstrained non-linear optimizer.

GGOPT is a derivative-free non-linear optimizer for smooth functions with added noise. If the function values arise from observations or from extensive computations, these errors can be considerable. GGOPT uses an adjustable mesh together with linear least squares to find smoothed values of the function, gradient and Hessian at the center of the mesh. These values drive a descent method that estimates optimal parameters. The smoothed values usually result in increased accuracy.

Algorithms

Correlations between the activities of semen acid phosphatase and Ca(2+)-dependent ATPase and age in different breeds of cocks.

1. The present work aims to find a biochemical criterion for evaluating the evolution of sperm according to age through the study of the ATPase activity from the spermatozoa and the acid phosphatase from the seminal plasma of cocks from three different breeds. 2. The optimal parameters of action of the cock semen acid phosphatase and the Ca(2+)-dependent ATPase from the spermatozoa were studied. 3. The substrate specificity of the semen acid phosphatase and its inhibition by tartrate, fluoride, metavanadate, molybdate and Hg2+ were also studied. 4. The two enzymes were determined from the Sussex, Golden Cornish and Plymouth Rock breeds at different ages. 5. The data lead to the conclusion that some properties of bird spermatozoa are less influenced by breed while the acid phosphatase activity, secreted in the ductus deferens is a breed characteristic.

Acid Phosphatase

Radiobiological speculations on therapeutic total body irradiation.

Unexpected total body irradiation (TBI) of human beings, involved in nuclear warfare or in accidents in nuclear reactors can be lethal. In the 1950s, bone marrow transplantation was discovered as a potentially life saving procedure after TBI in the dose range of 5.0 to 12.0 Gy. Since that time, deliberate or "therapeutic" TBI has been used to condition patients with a lethal bone marrow disorder for bone marrow replacement. The therapeutic ratio of TBI followed by bone marrow transplantation is small. Many potentially lethal complications can occur, such as acute TBI side effects, late TBI side effects or immunological complications of bone marrow transplantation such as graft versus host disease or graft rejection. The benefits of TBI and bone marrow transplantation are that they offer a chance for cure of previously lethal bone marrow disorders. The optimal parameters for TBI remain to be defined. The review discusses the current clinical and experimental animal data, as they relate to the future definition of less toxic TBI procedures with a better therapeutic ratio. Different TBI procedures are required for patients with malignant vs. non-malignant disorders or for patients with histoincompatible vs. histocompatible bone marrow donors.

Animals

Technical note: reconstructing dose distributions from manually planned electron boosts in breast radiotherapy.

PURPOSE: In breast radiotherapy, delivery of manually-calculated electron boosts limits retrospective dose-response analyses as dose distribution is unavailable. This work evaluates the feasibility of reconstructing dose distributions from manually planned electron boosts in breast-conserving radiotherapy. METHODS: Only 72 out of 198 breast cancer patients had complete stored dose distributions from sequential electron boosts in the REQUITE study. Arbitrary data from 70/72 patients were used to develop and validate dose reconstruction method. Twenty patients were used to determine optimal parameters for Monte-Carlo-based (MC) electron dose reconstruction on RayStation (v.11B-R), considering CT-calibration curve, MC-history number, andcalculation grid resolution. Remaining 50 patients were used to quantify dose reconstruction accuracy. The similarity between reconstructed and stored dose was evaluated using 3D-gamma index and dosimetric parameters extracted from breast and tumour bed contours. Dose difference location was evaluated using dose-location histogram. RESULTS: Calculation grid resolution significantly impacted electron dose distribution (p&#xa0;<&#xa0;0.01), where the finest grid (0.15&#xa0;cm) showed highest similarity to stored doses. CT-calibration curve and MC-history number had a negligible influence on dose reconstruction. Dosimetric difference between reconstructed and stored doses was&#xa0;<&#xa0;1&#xa0;Gy for breast and tumour bed. Reconstructed dose was achieved&#xa0;>&#xa0;90% gamma passing rate in the validation set. However, around 2.5&#xa0;Gy dose differences were observed at the skin and tissue interface regions. CONCLUSIONS: Retrospective electron boost dose reconstruction is feasible with acceptable accuracy, and could increase data completeness in large cohort studies. Caution is advised when assessing dose near tissue interface and further validation is needed outside the REQUITE dataset.

Electrons